Cytochrome p450 oxygenases

ABSTRACT

Nucleic acids encoding cytochrome P450 variants are provided. The cytochrome P450 variants of have a higher alkane-oxidation capability, alkene-oxidation capability, and/or a higher organic-solvent resistance than the corresponding wild-type or parent cytochrome P450 enzyme. A preferred wild-type cytochrome P450 is cytochrome P450 BM-3. Preferred cytochrome P450 variants include those having an improved capability to hydroxylate alkanes and epoxidate alkenes comprising less than 8 carbons, and have amino acid substitutions corresponding to V78A, H236Q, and E252G of cytochrome P450 BM-3. Preferred cytochrome P450 variants also include those having an improved hydroxylation activity in solutions comprising co-solvents such as DMSO and THF, and have amino acid substitutions corresponding to T235A, R471A, E494K, and S1024E of cytochrome P450 BM-3.

This application claims priority from U.S. Provisional Application Ser.No. 60/306,766, filed Jul. 20, 2001, and U.S. Provisional ApplicationSer. No. 60/308,429, filed Jul. 27, 2001. Each of these priorapplications is hereby incorporated by reference in its entirety.

The Government has certain rights to this invention pursuant to GrantNos. DBI-9807460 (ETF) and BES-9981770 (FHA) awarded by the NationalScience Foundation.

FIELD OF THE INVENTION

The invention relates to novel uses for cytochrome P450 BM-3. Inaddition, the invention relates to novel variants of cytochrome P450enzymes which are more resistant to organic solvents and/or are capableof oxidizing alkanes or alkenes.

BACKGROUND

The term paraffin (from Latin parum affinis=slight affinity) accuratelyreflects the nature of alkanes: these compounds are notoriously inert,and activating their C—H bonds presents a difficult chemical obstacle.In fact, one of the great challenges of contemporary catalysis is thecontrolled oxidation of hydrocarbons (Shilov 1997). Processes forcontrolled, stereo- and regioselective oxidation of hydrocarbon feedstocks to more valuable and useful products such as alcohols, ketones,acids, and peroxides would have a major impact on the chemical andpharmaceutical industries. However, selective oxyfunctionalization ofhydrocarbons thus remains one of the great challenges for contemporarychemistry. Despite decades of effort, including recent advances (Chen etal., 2000; Hartman and Ernst, 2000; Thomas et al., 2001), chemicalcatalysts for alkane functionalization are characterized by low yields,poor selectivity and harsh conditions.

Biocatalysts (enzymes) that oxidize alkanes allow organisms to utilizehydrocarbons as a source of energy and cellular building blocks (Ashrafet al., 1994; Watkinson and Morgan, 1990). Enzymes have uniqueproperties that distinguish them from most chemical catalysts. Mostimpressive is their ability to catalyze specific, and often difficult,chemical reactions in water at room temperature and atmosphericpressure. Forty years of screening alkane-assimilating organisms(Leadbetter and Foster, 1959) have identified a variety ofmulti-subunit, membrane-associated enzyme complexes, which have inspiredcuriousity and mimicry for their ability to catalyze selectiveoxidations at room temperature and ambient pressure (Scheller et al.,1996; Stevenson et al., 1996; Fox et al., 1990; Fisher et al., 1998;Benson et al., 1979). However, low catalyst turnover rates and limitedstability make applications of biocatalytic C—H bond activation feasibleonly in a very few industrial processes where high value compounds areproduced (Schmid et al., 2001).

Monooxygenases have unique properties that distinguish them from mostchemical catalysts. Most impressive is their ability to catalyze thespecific hydroxylation of non-activated C—H; one of the most usefulbiotransformation reactions, which is often difficult to achieve bychemical means, especially in water, at room temperature underatmospheric pressure. However, for chemical synthesis, organic solvents,not aqueous solutions, are generally used. The use of organic solventshas many advantages, most importantly are a) higher solubility of oftenin aqueous solutions poor soluble nonpolar compounds; b) suppression ofwater-dependent side reactions; c) alteration in enantioselectivity; andd) elimination of microbial contaminations (Dordick, 1992). The maindrawback of enzymes functioning in organic solvents is their drasticallyreduced catalytic activity caused by dehydration of the enzyme(Klibanov, 1997). Little is known about this process and mainlyhydrolytic enzymes such as esterases and lipases were used to study andimprove their activity and stability in organic solvents (Kvittingen etal., 1992). Cofactor dependent oxidative enzymes have multiple domainsand highly regulated electron transfer mechanisms to transport thereduction equivalent to the catalytic heme center (Munro et al., 1996;Beratan, 1996; Moser et al., 1995). Organic solvents can interfere byaffecting redox potentials and interactions between single domains.However, no theory has been developed to explain the influence oforganic solvents toward complex oxidative enzymes. Thus, the low organicsolvent resistance of enzymes, in particular enzymes suitable foroxidation of hydrophobic substances, is a particularly challengingproblem.

Cytochrome P450 Monoxygenases

The cytochrome P450 monooxygenases (“P450s”) is a group ofwidely-distributed heme enzymes that inserts one oxygen atom of O₂ intoa diverse range of hydrophobic substrates, often with high regio- andstereoselectivity. The second oxygen atom is reduced to H2O. The activesites of all cytochrome P450s contain an iron protoporphryin IX withcysteinate as the fifth ligand; the final coordination site is left tobind and activate molecular oxygen (Groves et al., 1995). For many ofthe P450-catalyzed reactions, no chemical catalysts come close inperformance (Lewis, 1996). These enzymes, however, are often only poorlyactive towards nonnatural substrates and cannot tolerate normal processconditions, including organic solvents (Lewis, 1996; Kuhn-Velten, 1997).Simply put, they are a process engineering nightmare.

One particular P450 enzyme, cytochrome P450 BM-3 from Bacillusmegaterium (EC 1.14.14.1) also known as CYP102, is a water-soluble,catalytically self-sufficient P450 containing a monooxygenase domain (64kD) and a reductase domain (54 kD) in a single polypeptide chain (Narhiand Fulco, 1986 and 1987; Miura and Fulco, 1975; Ruettinger et al.,1989). The minimum requirements for activity are substrate, dioxygen andthe cofactor nicotinamide adenine dinucleotide phosphate (NADPH).Nucleotide and amino acid sequences for P450 BM-3 can be found in, andare hereby incorporated by reference from, the GenBank database underthe accession Nos. J04832 (SEQ ID NO:1) and P14779 (SEQ ID NO:2),respectively.

P450 BM-3 hydroxylates fatty acids of chain length between C12 and C18at subterminal positions, and the regioselectivity of oxygen insertiondepends on the chain length (Miura and Fulco, 1975; Boddupalli et al.,1990). The optimal chain length of saturated fatty acids for P450 BM-3is 14-16 carbons, and the enzyme was initially believed to have noactivity towards fatty acids smaller than C12 (Miura and Fulco, 1975).P450 BM-3 is also known to hydroxylate the corresponding fatty acidamides and alcohols and forms epoxides from unsaturated fatty acids(Miura and Fulco, 1975; Capdevila et al., 1996; Graham-Lorence et al.,1997; Ruettinger and Fulco, 1981). The enzyme was reported to beinactive towards alkanes and methyl esters lacking the polarfunctionality of the natural substrates (Miura and Fulco, 1975).However, there were indications that P450 BM-3 could acceptshorter-chain alkanes, although with very low activity (Munro et al.,1993).

Powerful techniques for creating enzymes with modified or improvedproperties are now available, such as directed evolution (Arnold, 1998),in which iterative cycles of random mutagenesis, recombination andfunctional screening for improved enzymes accumulate the mutations thatconfer the desired properties. Mutant P450 BM-3 enzymes with modifiedactivity have now been reported in the literature. For example, an F87A(Phe87→Ala87) mutant was found to display a higher activity for the12-pCNA substrate (Farinas et al., 2001; Schwaneberg et al., 1999). Inaddition, by reengineering the fatty acid binding site to accommodatefatty acids with a chain-length shorter than 12 (Li et al., 2001; Ost etal., 2000), Li and coworkers (2001) found mutants that are capable tohydroxylate indole which dimerizes in the presence of oxygen to indigoand indinlbin, and Carmichael and Wong (2001) found P450 BM-3 mutantsthat could oxidize polycyclic aromatic hydrocarbons (“PAHs”) such asphenanthrene, fluoranthene, and pyrene. In addition, the Schmid grouprecently reported mutants of P450 BM-3 that can hydroxylate a variety ofnonnatural substrates, including octane, several aromatic compounds andheterocyclic compounds (Appel et al., 2001). In addition, P450 BM-3mutants for epoxidation of substrates such as long-chain unsaturatedfatty acids (Miura and Fulco, 1975; Capdevila et al., 1996;Graham-Lorence et al., 1997; Ruettinger and Fulco, 1981) and styrene(Fruetel, J A et al., 1994) have been suggested.

Many of the wild-type and mutant P450 BM-3 substrates have, if theperformance of the catalyst is sufficient, bright prospectiveapplications as products or intermediates in fine chemical synthesis(Schneider et al., 1999). Unfortunately, until now, the alkanehydroxylation activities of P450 BM-3 mutants are still limited,especially for lower alkanes. Moreover, many of P450 BM-3 substrates(fatty acids, alcohols, amides, C>12 alkanes, polyaromatic hydrocarbons,heterocycles, etc.), are notoriously insoluble in aqueous solution andrequire for solubilization an organic co-solvent, and the organicsolvent resistance of P450 BM-3 in water miscible co-solvents is low andinsufficient for industrial applications. To date, no P450 BM-3 mutantswith improved or altered solvent resistance have been identified.

Thus, there is a need in the art for industrially useful oxidationcatalysts for alkane hydroxylation and alkene epoxidation; particularlyon hydrocarbon substrates that are shorter than its preferredsubstrates, the fatty acids. While various alkane hydroxylases areknown, for example w-hydroxylase and methane monooxygenase, none ofthese naturally-occurring enzymes have the practical advantages of anenzyme such as P450 BM-3, which is highly expressed in recombinant formin bacteria and contains all its functional domains in a singlepolypeptide chain. There is also a need for oxygenase enzymes that canoperate efficiently in organic co-solvents. This invention addressesthese and other needs in the art.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of P450 BM-3 variantsthat have improved alkane oxidation activity, alkene oxidation activity,and/or improved stability in organic co-solvents.

Accordingly, the invention provides an isolated nucleic acid encoding acytochrome P450 variant which has a higher capability than thecorresponding wild-type cytochrome P450 to oxidize at least onesubstrate selected from an alkane comprising a carbon-chain of no morethan 8 carbons and an alkene comprising a carbon-chain of no more than 8carbons, wherein the wild-type cytochrome P450 comprises an amino acidsequence having at least 60% sequence identity to SEQ ID NO:2 (i.e., thewild-type sequence of P450 BM-3), and the cytochrome P450 variantcomprises an amino acid substitution at a residue corresponding to aresidue of SEQ ID NO:2 selected from V78, H236, and E252. In oneembodiment, the amino acid sequence has at least 80% sequence identityto SEQ ID NO:2. Preferably, the amino acid sequence is SEQ ID NO:2. Inanother embodiment, the higher capability is a higher maximum turnoverrate of the substrate into an oxidized product, and the maximum turnoverrate of the variant is at least 5 times, preferably at least 10 times,the maximum turnover rate of the wild-type. When the substrate is analkane, the capability to oxidize is preferably the capability tohydroxylate, and the alkane can be selected from one or more of octane,hexane, cyclohexane, pentane, butane, and propane. When the substrate isan alkene, the capability to oxidize can be either or both of thecapability to epoxidate or hydroxylate, and the alkene can be selectedfrom one or more of 1-hexene, 2-hexene, 3-hexene, cyclohexene, isoprene,allyl chloride, propene, and styrene.

In another embodiment, the cytochrome P450 variant comprises amino acidsubstitutions at residues corresponding to at least two residues of SEQID NO:2 selected from V78, H236, and E252, preferably at all of theseresidues, and, even more preferably, the amino acid substitution isselected from V78A, H236Q, and E252G. When the wild-type sequence isthat of P450 BM-3, the cytochrome P450 variant can comprise amino acidsubstitutions at two residues selected from V78, H236, and E252,preferably all of these residues, and, even more preferably, the aminoacid substitutions are V78A, H236Q, and E252G. In addition, thecytochrome P450 variant may comprise at least one further amino acidsubstitution at a residue selected from H138, T175, V178, A184, N186,F205, D217, S226, R255, A290, A295, L353, and G396. Optionally, theamino acid substitution at these residues is selected from one or moreof H138Y, T175I, V178I, A184V, N186D, F205C, D217V, S226I, S226R, R255S,A290V, A295T, L353V, and G396M. In preferred embodiments, the cytochromeP450 variant comprises the amino acid substitutions V78A, H138Y, T175I,V178I, A184V, H236Q, E252G, R255S, A290V, A295T, and L353V, or the aminoacid substitutions V78A, T175I, A184V, F205C, S226R, H236Q, E252G,12255S, A290V, and L353V.

The invention also provides an isolated nucleic acid encoding acytochrome P450 variant which has a higher organic solvent-resistancethan the corresponding wild-type cytochrome P450, wherein the wild-typecytochrome P450 comprises an amino acid sequence has at least 60%sequence identity to SEQ ID NO:2, and the cytochrome P450 variantcomprises an amino acid substitution at a residue corresponding to aresidue of SEQ ID NO:2 selected from T235, R471, E494, and S1024. In oneembodiment, the amino acid sequence has at least 80% sequence identityto SEQ ID NO:2. Preferably, the amino acid sequence is SEQ ID NO:2. Theorganic solvent-resistance may be, for example, a higher maximumturnover rate of a substrate into an oxidized product in a solutioncomprising an organic solvent, and the oxidized product may be ahydroxylated product. In another embodiment, the organic solvent isselected from THF, DMSO, methanol, ethanol, propanol, dioxane, anddimethylformamide. For example, when the solution comprises 25% (v/v)DMSO, the hydroxylation activity of the cytochrome P450 BM-3 variant canbe at least twice the hydroxylation activity of wild-type cytochromeP450 BM-3. Also, when the solution comprises 2% (v/v) THF, thehydroxylation activity of the cytochrome P450 BM-3 variant can be atleast twice the hydroxylation activity of wild-type cytochrome P450BM-3.

In particular embodiments, the cytochrome P450 variant may compriseamino acid substitutions at residues corresponding to at least tworesidues of SEQ ID NO:2 selected from T235, R471, E494, and S1024;preferably at least three residues, an the amino acid substitutions atresidues may correspond to T235, R471, E494, and S1024 of SEQ ID NO:2.Alternatively, when the wild-type sequence is SEQ ID NO:2, thecytochrome P450 variant may comprise amino acid substitutions at leasttwo residues selected from T235, R471, E494, and S1024, preferablythree, and, even more preferably, the cytochrome P450 variant comprisesamino acid substitutions at T235, R471, E494, and S1024. Optionally, theamino acid substitutions can be T235A, R471A, E494K, and S1024E. In anyof the foregoing embodiment, the cytochrome P450 variant further mayfurther comprise an amino acid substitution at residue F87, for example,F87A. In a preferred embodiment, the cytochrome P450 variant comprisesthe mutations F87A, T235A, R471A, E494K, and S1024E.

The invention also provides for an isolated nucleic acid encoding avariant of a parent cytochrome P450 oxygenase, the variant having (i) ahigher ability than the parent to oxidize a substrate selected from analkane comprising a carbon-chain of no more than 8 carbons or alkenecomprising a carbon-chain of less than 8 carbons; and (ii) at least oneamino acid substitution in a secondary structural element of thecytochrome P450 heme domain selected from the helix B′ domain, the helixH domain, and the helix I domain, wherein the parent comprises an aminoacid sequence having at least 60% sequence identity to SEQ ID NO:2.Preferably, the amino acid sequence of the parent has at least 80%sequence identity to SEQ ID NO:2. Even more preferably, the amino acidsequence of the parent is SEQ ID NO:2. The amino acid substitution can,for example, be at a residue corresponding to a residue of SEQ ID NO:2selected from V78, H236, and E252, and correspond to V78A, H236Q, orE252G. Optionally, the variant further comprises at least one amino acidsubstitution at a residue corresponding to a residue of SEQ ID NO:2selected from 11138, T175, V178, A184, F205, S226, R255, A290, A295, andL353, such as, for example, V78A, H138Y, T175I, V178I, A184V, F205C,S226R, H236Q, E252G, R255S, A290V, A295T, and L353V. Preferably, theamino acid substitutions are at residues corresponding to amino acidresidues V78, H236, and E252 of SEQ ID NO:2, such as, for example, V78A,H236Q, and E252G.

The invention also provides for an isolated nucleic acid encoding avariant of a parent cytochrome P450 oxygenase, the variant having: (i) ahigher organic solvent resistance than the parent; and (ii) at least oneamino acid substitution in a secondary structural element of theselected from the helix H domain of the cytochrome P450 heme domain andthe helix of the flavin domain; wherein the parent comprises an aminoacid sequence having at least 60% sequence identity to SEQ ID NO:2. Inone embodiment, the amino acid sequence of the parent has at least 80%sequence identity to SEQ ID NO:2. Preferably, the amino acid sequence ofthe parent is SEQ ID NO:2. The amino acid substitution can be at aresidue corresponding to a residue of SEQ ID NO:2 selected from T235 andE494, and the cytochrome P450 variant may optionally comprise a furtheramino acid substitution at a residue corresponding to a residue of SEQID NO:2 selected from R471 and S1024. If so, the amino acid substitutioncan be selected from T235A and E494K, and the further amino acidsubstitution can be selected from R471A and S1024E. In a preferredembodiment, the amino acid substitution is selected from T235A andE494K, and the further amino acid substitution is selected from R471Aand S1024E. In another preferred embodiment, the variant furthercomprises an amino acid substitution at a residue corresponding toresidue F87 of SEQ ID NO:2, such as, for example, F87A, F87G, F87V,F87I, F87F, F87W, F87D, F87N, F87H, F87K, or F87R. A preferred variantcomprises amino acid substitutions at residues corresponding to aminoacid residues T235, R471, E494, and S1024 of SEQ ID NO:2, and the aminoacid substitutions are preferably T235A, R471A, E494K, and S1024E, withor without the amino acid substitution F87A.

The invention provides for an isolated nucleic acid encoding acytochrome P450 variant, the cytochrome P450 variant comprising theamino acid substitutions V78A, H236Q, and E252G of SEQ ID NO:2, whereinthe variant may further comprise the amino acid substitutions H138Y,T175I, V178I, A184V, N186D, D217V, S226I, R255S, A290V, A295T, L353V,and G396M, or the ammo acid substitutions T175I, A184V, F205C, S226R,R255S, A290V, L353V.

The invention also provides for an isolated nucleic acid encoding acytochrome P450 variant, the cytochrome P450 variant comprising theamino acid substitutions T235A, R471A, E494K, and S1024E of SEQ ID NO2,optionally comprising the amino acid substitution F87A.

Finally, the invention also provides for amino acid sequences comprisingthe above-mentioned amino acid substitutions.

The above features and many other advantages of the invention willbecome better understood by reference to the following detaileddescription when taken in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Gas chromatogram of the oxidation products of octane, catalyzedby wild-type P450 BM-3.

FIG. 2. The screening assay for alkane oxidation activity uses thesubstrate analog 8-pnpane (p-nitrophenoxyoctane). Terminal hydroxylationgenerates the unstable hemiacetal, which decomposes to the aldehyde andp-nitrophenolate, which is monitored at 410 nm.

FIG. 3. P450 BM-3 screening procedure. Library of P450 BM-3 mutant genesis transformed into E. coli and plated on agar, from which singlecolonies are picked into 96-well plates and grown overnight. From theseplates, samples are taken to inoculate fresh 96-well plates, in whichthe enzymes are expressed and assayed for hydroxylation activity. Theplates from the overnight growth are stored at 4° C. and used to isolateactive clones identified in the assay.

FIG. 4. Progression of activity for P450 BM-3, as measured on 8-pnpane.

FIG. 5. Initial rates of NADPH oxidation vs alkane concentration forpurified P450 BM-3 mutant IX 139-3. Octane: k_(cat)=35.6 s⁻¹,Km=9.1×10⁻⁴ M and k_(cat)/K_(m)=3.9×10⁴ M⁻¹s⁻¹. Hexane: k_(cat)=34.3s⁻¹, K_(m)=4.9×10⁻⁴ M and k_(cat)/K_(m)=7.0×10⁴ M⁻¹s⁻¹. Cyclohexane:k_(cat)=31.0 s⁻¹, K_(m)=8.4×10⁻⁵ M and k_(cat)/K_(m)=3.7×10⁵ M⁻¹s⁻¹.

FIG. 6. Gas chromatogram of the oxidation products of hexane, catalyzedby IX79_(—)1.

FIG. 7. Gas chromatogram of the oxidation products of cyclohexane,catalyzed by IX79_(—)1.

FIG. 8. Gas chromatogram of the oxidation products of butane, catalyzedby IX139-3.

FIG. 9. Alternative representation of the pNCA assay principle (see alsoFIG. 2).

FIG. 10. Re-screen results of 1st mutant generation, investigatingorganic solvent resistance in 1% (v/v) (see Example 2).

FIGS. 11A and B. Mutagenic pathways for parent F87A and the co-solventsDMSO (A) and THF (B) (see Example 2), showing increased residualactivity versus enzyme generation.

FIGS. 12A to C. Organic solvent resistance profile of the parent,showing organic solvent resistance against percentage of solvent (seeExample 2). (A) DMSO as solvent. (B) THF as solvent. (C) Purifiedwild-type, F87A, and F87ASB3, using DMSO as solvent.

FIGS. 13A to F. Organic solvent resistance profile of back-mutated BM-3variants. Solvent: A=DMSO; B=THF; C=acetone; D=acetonitrile; E=DMF; andF=ethanol.

FIG. 14. Maximum turnover rates (mole substrate/min/mole enzyme) forP450 BM-3 wildtype (black bars) and IX139-3 (shaded bars) on alkane andfatty acid substrates.

FIG. 15. Maximum rates reported for alkane hydroxylation by alkanemonooxygenases CYP4B111, CYP52A38, P450cam9, AlkB18, and sMMO10. Ratesfor P450 BM-3 wildtype and mutant IX139-3 were determined in this work.

FIGS. 16A and B. Optical spectra for IX139-3 and wildtype P450 BM-3. (A)Mutant IX139-3 (0.5 mM) in potassium phosphate buffer (0.1 M, pH 8)(-▴-); with laurate (0.5 mM, solid line) in 1% methanol; hexane (1.0 mM,-▪-) in 1% methanol; propane (saturated solution, --). (B) WildtypeP450 BM-3 in potassium phosphate buffer (0.1 M, pH 8) (-▴-); withlaurate (0.5 mM, solid line) in 1% methanol; hexane (1.0 mM, 1.0 mM,—▪—) in 1% methanol; propane (saturated solution, --).

FIG. 17. Initial rates of NADPH consumption (mole substrate/min/moleenzyme) in the presence of alkenes for wild-type (black bars) andIX139-3 (shaded bars). “ND” indicates that NAPDH consumption was notdetectable over background.

FIG. 18. Gas chromatogram of the oxidation products of styrene (A) andpropene (B) catalyzed by IX139-3.

FIGS. 19A and B. (A) Absorption spectra in the presence of IX139-3 afterthe 4-NBP assay with isoprene and NADPH (o), NADPH without isoprene (□),and isoprene without NADPH (Δ). (B) Absorption spectra in the presenceof IX139-3 after the NBP assay with styrene and NADPH (o), NADPH withoutstyrene (□), and styrene without NADPH (Δ).

FIGS. 20A and B. Sequence alignments of P450 BM-3 (GenBank Accession No.P14779 (SEQ ID NO:2)) with CYP 2C3 (GenBank P00182, SEQ ID NO:3), CYP2C9 (GenBank P11712; SEQ ID NO:4), CYP 2D1v (GenBank P10633; SEQ IDNO:5), and CYP 108 (GenBank P33006; SEQ ID NO:6).

FIGS. 21A to F. Representative topology diagrams of the heme domain ofP450 variants of the invention, based on P450BM-P; the heme domain ofP450 BM-3. (A) topology of P450BM-P; the topology is depicted withhelices represented by black bars, and the length of each of the bars isin approximate proportion to the length of the helix. The strands ofβ-sheets are shown with arrows. The strands are grouped by the secondarystructural elements which they comprise. The structural elements aregrouped into the α-helical-rich domain and the β-sheet-residues H138 andA78 relative to the heme group. (D) Location of residues T175, V178,A184, 1995). (B) Topology of P450 BM-3, showing location of the hemegroup. (C) Location of rich domain. The heme is shown by the square atthe NH₂-terminal end of the L-helix. With only minor modifications, thistopology diagram could be used for other P450s (Peterson et al., N186,and D217 relative to the heme. (E) Location of residues H236, R255, andE252 relative to the heme. (F) Location of residues L353, G396, A290,and A295 relative to the heme. Those sections labeled A, beta1, beta3,beta4, D, E, F, G, I, K, and J denote secondary structural elementsconserved in P450s.

FIG. 22. Positions of the amino acid substitutions in P450 BM-3 mutantIX139-3. The variant contains 11 amino acid substitutions, which arerepresented as spheres on the crystal structure of the substrate-boundenzyme (PDB: 1FAG). Five are clustered on the highly flexible F-Ghelix-loop-helix structure and the I helix along which it slides duringsubstrate binding and release.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides novel variants or mutants of P450 BM-3 which arecapable of oxidizing alkanes, alkane derivatives, and/or saturated orunsaturated hydrocarbons. Accordingly, the invention provides variantP450 BM-3 enzymes which have a higher oxidation activity towards atleast one alkane, alkane derivative, alkene, or alkene derivative thanwild-type P450 BM-3. Preferably, the variant P4590 BM-3 has a higheroxidation activity towards a saturated hydrocarbon such as octane,hexane, cyclohexane, propane, ethane, and/or butane, or towards anunsaturated hydrocarbon such as propene, hexene, cyclohexene, isoprene,allyl chloride, and/or styrene, or derivatives thereof, than wild-typeP450 BM-3. In one embodiment, the P450 BM-3 variants comprise mutationsat one or more of the following residues of SEQ ID NO:2, counting thestarting methionine as position 0 (“zero”): V78, H138, T175, V178, A184,N186, F205, D217, S226, H236, E252, 8255, A290, A295, L353, and G396.Preferably, the mutation or mutations are selected from V78A, H138Y,T175I, V178I, A184V, N186D, F205C, D217V, S226I, S226R, H236Q, E252G,R255S, A290V, A295T, L353V, and G396M. The P450 BM-3 variants cancomprise at least one, preferably at least three, and more preferably atleast 5, and even more preferably at least all of these amino acidmutations. In a particularly preferred embodiment, the P450 BM-3 variantcomprises the mutations V78A, H236Q, and the E252G mutations. See alsoTable 1A.

In addition, the invention provides P450 BM-3 variants with a higherorganic solvent resistance in at least one water-miscible co-solventthan wild-type P450 BM-3. Preferably, the P450 BM-3 variants of theinvention have a higher organic solvent resistance towardswater-miscible co-solvents such as, but not limited to, DMSO, THF,methanol, ethanol, propanol, dioxane, and dimethylfonamide. In apreferred embodiment, the P450 BM-3 variant comprises mutations at oneor more of the following residues of SEQ ID NO:2, counting the startingmethionine as position 0 (“zero”): F87, T235, R471, E494, and S1024. Themutation at F87 can be, for example, F87A, F87G, F87V, F871, F87F, F87W,F87D, F87N, F87H, F87K, F87R. The mutations at T235, R471, and E494 canbe, for example, T235A, R471C, R471A, and E494K. The mutation at S1024can be, for example, S1024R, S1024T, S1024K, and S1024E. In a preferredembodiment, the novel P450 BM-3 variants comprise mutations in at leastone, preferably at least three of amino acid residues F87, T235, R471,E494, and S1024. Most preferably, the variant comprises the mutationsT235A, R471A, E494K, and S1024E, with or without the mutation F87A. Seealso Table 1A.

TABLE 1A Preferred Cytochrome P450 Mutated Amino Acid Residues andMutations Amino Acid Residue of SEQ ID NO: 2 Amino Acid Mutation V78V78A F87 F87A H138 H138Y T175 T175I V178 V178I A184 A184V N186 N186DF205 F205C D217 D217V S226 S226I, S226R T235 T235A H236 H236Q E252 E252GR255 R255S A290 A290V A295 A295T L353 L353V G396 G396M R471 R471A E494E494K S1024 S1024E

In addition, the invention provides for P450 BM-3 mutants havingspecific nucleic acid and amino acid sequences. The nucleic acidsequences include those which encode for the P450 BM-3 variants in Table1B. The amino acid sequences include those which have the combinationsof amino acid mutations in Table 1B, where all mutations refer to SEQ IDNO:2, counting the starting methionine residue as position 0 (“zero”).

TABLE 1B Preferred P450 BM-3 Variants Amino Acid Mutations in Wild-TypeP450 BM-3 Designation (SEQ ID NO: 2) ix139-3 V78A, H138Y, T175I, V178I,A184V, H236Q, E252G, R255S, A290V, A295T, L353V ix139-37 V78A, T175I,A184V, N186D, D217V, H236Q, E252G, A290V, L353V, G396M ix139_43J V78A,T175I, A184V, S226I, H236Q, E252G, A290V, L353V V78A, T175I, A184V,F205C, S226R, H236Q, E252G, R255S, A290V, L353V F87A5F5 F87A, T235A,R471A, E494K, S1024E F87ABC1F10 F87A, T235A, R471A, S1024T W5F5 T235A,R471A, E494K, S1024E

Moreover, the invention provides for novel variants of P450 enzymesother than P450 BM-3, which have a higher activity for alkane oxidationand/or higher solvent resistance than the corresponding wild-typeenzyme. These novel variants can be identified when aligned with therespective non-BM-3 amino acid sequence with that of P450 BM-3 (SEQ IDNO:2), amino acid positions in the non-BM-3 sequence that are alignedwith one or more of the following amino acid residues in SEQ ID NO:2 areidentified: V78, F87, H138, T175, V178, A184, N186, F205, D217, S226,T235, H236, E252, R255, A290, A295, L353, G396M, R471, E494, and S1024.Mutations in amino acid residues of the non-BM-3 enzyme which arealigned with and identical to the aforementioned BM-3 amino acidresidues results in novel P450 variants according to the invention. SeeFIG. 20. Preferably, the mutation in the non-BM-3 sequence results inone or more of the following amino acid substitutions: V78′A, F87′A,F87′G, F87′V, F87′1, F87′F, F87′W, F87′D, F87′N, F87′H, F87′K, F87′R,H138′Y, T175′1, V178I, A184′V, N186′D, F205′C, D217′V, S226′I, S226′R,T235′A, H236′Q, E252′G, R255′S, A290′V, A295′T, L353′V, G396′M, R471′C,R471′A, E494′K, S1024′R, S1024′T, S1024′K, and S1024′E, where the aminoacid residue is the amino acid residue aligned with the correspondingP450-BM-3 residue (denoted by prime (′) sign). Preferred, non-limitingexamples of such novel “non-BM-3” enzymes are listed in Table 2.

TABLE 2 Preferred Non-P450 BM-3 Variants Wild-Type Aligned Amino P450BM-3 Wild-Type Enzyme Acid Sequence Mutation(s) Mutation(s) CYP 2C3 SEQID NO: 3 H266Q H236′Q CYP2C9 SEQ ID NO: 4 E285G E252′G CYP 2D1v SEQ IDNO: 5 E296G, L398V E252′G, L353′V CYP 108 SEQ ID NO: 6 A293V A290′V

In addition, the invention provides for variants of non-BM-3 enzymes,wherein the wild-type sequences are at least 30, preferably at least 50,more preferably at least 70, even more preferably at least 90%, andoptimally at least 95% identical to SEQ ID NO: 2. Preferred,non-limiting examples of such novel “non-BM-3 P450s” and their hemedomains are described herein, listed in Table 2 and depicted in FIG. 20.In one embodiment, the oxidase activity of a P450 variant for one ormore alkane- or alkene-substrates is at least five, more preferably atleast 10, and even more preferably at least 15 times that of thecorresponding wild-type cytochrome P450. In another embodiment, theorganic solvent resistance of a P450 variant is at least two, preferablyat least 3, and even more preferably at least five times that of thecorresponding wild-type cytochrome P450.

In addition, the “non-BM-3 P450” may be a P450 BM-3 variant, which hasone or more mutations as compared to wild-type P450 BM-3. Such variants,including variants displaying more than 60% sequence identity to SEQ IDNO:2, are described in, e.g., PCT application PCT/US02/11954, filed Apr.16, 2002.

Wild-Type Cytochrome P450 Enzymes

Crystal structures of wildtype P450 BM-3 with and without substratereveal large conformational changes upon substrate binding at the activesite (Haines et al., 2001; Li and Poulos, 1997; Paulsen and Ornstein,1995; and Chang and Loew, 2000). The substrate free structure displaysan open access channel with 17 to 21 ordered water molecules. Substraterecognition serves as a conformational trigger to close the channel,which dehydrates the active site, increases the redox potential, andallows dioxygen to bind to the heme.

The activity of P450 BM-3 on saturated fatty acids follows the orderC15=C16>C14>C17>C13>C18>C12 (Oliver et al., 1997). On the C16 fattyacid, k_(cat)=81 s⁻¹ and K_(m)=1.4×10⁻⁶ M (k_(cat)/K_(m)=6.0×10⁷M⁻¹s⁻¹). With the C12 fatty acid, kcat=26 s⁻¹, K_(m)=136×10⁻⁶ M andk_(cat)/K_(m)=1.9×10⁵ M⁻¹s⁻¹ (Oliver et al., 1997).

P450 BM-3 may be compared to naturally-occurring enzyme thathydroxylates linear alkanes. For example, Pseudomonas oleovorans is ableto oxidize n-alkanes using hydroxylase machinery comprising an integralmembrane oxygenase (ω-hydroxylase), a soluble NADH-dependent reductaseand a soluble metalloprotein (rubredoxin) which transfers electrons fromthe reductase to the hydroxylase (Staijen et al., 2000). Theω-hydroxylase has been cloned from P. oleovorans into Escherichia coli,where it has been expressed and purified (Shanklin et al., 1997). Thespecific activity of this ω-hydroxylase for octane (5.2 units/mghydroxylase (Shanklin et al., 1997)) is ˜13 times greater than that ofP450 BM-3 (0.4 units/mg enzyme) (See Example 1). (The specific activityof the complete P. oleovorans system, including the rubredoxin and thereductase, is of course less than 5.2 units/mg). Thus, wildtype P450BM-3 is inefficient relative to this (and other) naturally occurringenzyme for alkane hydroxylation.

A tyrosine (Tyr51) at the entrance to the substrate-binding pocket makesa hydrogen bond to the carboxylate group of the substrate in the crystalstructure of the enzyme bound with palmitoleic acid (Li and Poulos,1997). Arg 47, also at the entrance to the binding pocket, may form anionic interaction as well. Nonpolar alkane substrates must rely solelyon hydrophobic partitioning into the enzyme's extended substratechannel, and poor substrate recognition may contribute to P450 BM-3'ssluggish activity on octane and other alkanes or alkenes.

Directed Evolution

The present invention provides evolved enzymes which oxidize alkanes toa higher degree, or which have a higher resistance to organic solventsand/or co-solvents, than the corresponding wild-type enzyme(s). Asdescribed in Example 3, a P450 BM-3 variant according to the inventionhas been generated which surpasses the activity of the alkanehydroxylase from P. oleovorans on octane. The mutant also showed similarhigh activity on hexane, cyclohexane, and pentane, which was not shownto be a substrate for P450 BM-3 before, and is also efficient on butaneand propane. Thus, the cytochrome P450 variants of the invention showthat it is possible to reach activities on unreactive nonnaturalsubstrates that are close to the activity of the native enzyme, e.g.,P450 BM-3, on its best natural substrates (for P450 BM-3; long chainfatty acids), which are about 1000 times higher than that of eukaryoticP450s and one of the highest activities of P450s known so far.

The strategy described here also provides improvements of the P450 BM-3activity on a number of substrates, including shorter chain alkenes suchas propene, allyl chloride, isoprene, 1-hexene and styrene. It shouldalso be possible to target other key properties such asregioselectivity, enantioselectivity and catalyst stability.

A preferred technique to improve the alkane-oxidation and co-solventresistance of wild-type or parent cytochrome P450 enzymes, includingP450 BM-3, is directed evolution. General methods for generatinglibraries and isolating and identifying improved proteins according tothe invention using directed evolution are described briefly below. Moreextensive descriptions can be found in, for example, Arnold (1998); U.S.Pat. Nos. 5,741,691; 5,811,238; 5,605,793 and 5,830,721; andInternational Applications WO 98/42832, WO 95/22625, WO 97/20078, WO95/41653 and WO 98/27230.

The basic steps in directed evolution are (1) the generation of mutantlibraries of polynucleotides from a parent or wild-type sequence; (2)(optional) expression of the mutant polynucleotides to create a mutantpolypeptide library; (3) screening/selecting the polynucleotide orpolypeptide library for a desired property of a polynucleotide orpolypeptide; and (4) selecting mutants which possess a higher level ofthe desired property; and (5) repeating steps (1) to (5) using theselected mutant(s) as parent(s) until one or more mutants displaying asufficient level of the desired activity have been obtained. Theproperty can be, but is not limited to, alkane oxidation capability andsolvent-resistance.

The parent protein or enzyme to be evolved can be a wild-type protein orenzyme, or a variant or mutant. The parent polynucleotide can beretrieved from any suitable commercial or non-commercial source. Theparent polynucleotide can correspond to a full-length gene or a partialgene, and may be of various lengths. Preferably the parentpolynucleotide is from 50 to 50,000 base pairs. It is contemplated thatentire vectors containing the nucleic acid encoding the parent proteinof interest may be used in the methods of this invention.

Any method can be used for generating mutations in the parentpolynucleotide sequence to provide a library of evolved polynucleotides,including error-prone polymerase chain reaction, cassette mutagenesis(in which the specific region optimized is replaced with a syntheticallymutagenized oligonucleotide), oligonucleotide-directed mutagenesis,parallel PCR (which uses a large number of different PCR reactions thatoccur in parallel in the same vessel, such that the product of onereaction primes the product of another reaction), random mutagenesis(e.g., by random fragmentation and reassembly of the fragments by mutualpriming); site-specific mutations (introduced into long sequences byrandom fragmentation of the template followed by reassembly of thefragments in the presence of mutagenic oligonucleotides); parallel PCR(e.g., recombination on a pool of DNA sequences); sexual PCR; andchemical mutagenesis (e.g., by sodium bisulfite, nitrous acid,hydroxylamine, hydrazine, formic acid, or by adding nitrosoguanidine,5-bromouracil, 2-aminopurine, and acridine to the PCR reaction in placeof the nucleotide precursor; or by adding intercalating agents such asproflavine, acriflavine, quinacrine); irradiation (X-rays or ultravioletlight, and/or subjecting the polynucleotide to propagation in a hostcell that is deficient in normal DNA damage repair function); or DNAshuffling (e.g., in vitro or in vivo homologous recombination of poolsof nucleic acid fragments or polynucleotides). Any one of thesetechniques can also be employed under low-fidelity polymerizationconditions to introduce a low level of point mutations randomly over along sequence, or to mutagenize a mixture of fragments of unknownsequence.

Once the evolved polynucleotide molecules are generated they can becloned into a suitable vector selected by the skilled artisan accordingto methods well known in the art. If a mixed population of the specificnucleic acid sequence is cloned into a vector it can be clonallyamplified by inserting each vector into a host cell and allowing thehost cell to amplify the vector and/or express the mutant or variantprotein or enzyme sequence. Any one of the well-known procedures forinserting expression vectors into a cell for expression of a givenpeptide or protein may be utilized. Suitable vectors include plasmidsand viruses, particularly those known to be compatible with host cellsthat express oxidation enzymes or oxygenases. E. coli is one exemplarypreferred host cell. Other exemplary cells include other bacterial cellssuch as Bacillus and Pseudomonas, archaebacteria, yeast cells such asSaccharomyces cerevisiae, insect cells and filamentous fungi such as anyspecies of Aspergillus cells. For some applications, plant, human,mammalian or other animal cells may be preferred. Suitable host cellsmay be transformed, transfected or infected as appropriate by anysuitable method including electroporation, CaCl₂ mediated DNA uptake,fungal infection, microinjection, microprojectile transformation, viralinfection, or other established methods.

The mixed population of polynucleotides or proteins may then be testedor screened to identify the recombinant polynucleotide or protein havinga higher level of the desired activity or property. Themutation/screening steps can then be repeated until the selectedmutant(s) display a sufficient level of the desired activity orproperty. Briefly, after the sufficient level has been achieved, eachselected protein or enzyme can be readily isolated and purified from theexpression system, or media, if secreted. It can then be subjected toassays designed to further test functional activity of the particularprotein or enzyme. Such experiments for various proteins are well knownin the art, and are described below and in the Examples below.

The evolved enzymes can be used in biocatalytic processes for, e.g.,alkane hydroxylation and alkene epoxidation, or for improving yield ofreactions involving oxidation of substrates with low solubility inaqueous solutions. The enzyme variants of the invention can be used inbiocatalytic processes for production of chemicals from hydrocarbons,particularly alkanes and alkenes, in soluble or immobilized form.Furthermore, the enzyme variants can be used in live cells or in deadcells, or it can be partially purified from the cells. One preferredprocess would be to use the enzyme variants in any of these forms(except live cells) in an organic solvent, in liquid or even gas phase,or for example in a super-critical fluid like CO₂. The organic solventwould dissolve high concentrations of the non-polar substrate, so thatthe enzyme could work efficiently on that substrate.

Recycling the cofactor can present difficulties for such a process.However, cofactor recycling methods well known in the art can beapplied. For example, an enzyme capable of regenerating the cofactor,using a second substrate can be used. Alternatively, the enzyme can beused in living cells, and the cofactor recycling can be accomplished byfeeding the cells the appropriate substrate(s). The NADPH and oxygen canalso be replaced by a peroxide, for example hydrogen peroxide, butylperoxide or cumene peroxide, or by another oxidant. Mutations thatenhance the efficiency of peroxide-based oxidation by BM-3 or othercytochrome P450 enzymes can serve to enhance the peroxide shunt activityof the enzyme variants described here. The mutations described here canbe combined with such mutations, for example, and tested for theircontributions to peroxide-driven alkane and alkene oxidation.

Screening Assays

The method of screening for identifying mutants or variants, for furthertesting or for the next round of mutation, will depend on the desiredproperty sought. For example, in this invention, recombinant nucleicacid which encode cytochrome P450 enzymes with improved alkane-oxidationcapability and/or solvent-resistance can be screened foralkane-oxidation activity or for activity or stability in asolvent/co-solvent mixture. Such tests are well known in the art.Examples of suitable tests are provided in the Examples and discussedbelow.

In a broad aspect, a screening method to detect oxidation comprisescombining, in any order, substrate, oxygen donor, and test oxidationenzyme. The assay components can be placed in or on any suitable medium,carrier or support, and are combined under predetermined conditions. Theconditions are chosen to facilitate, suit, promote, investigate or testthe oxidation of the substrate by the oxygen donor in the presence ofthe test enzyme, and may be modified during the assay. The amount ofoxidation product, i.e., oxidized substrate, is thereafter detectedusing a suitable method. Further, as described in WO 99/60096, ascreening method can comprise a coupling enzyme such as horseradishperoxidase to enable or enhance the detection of successful oxidation.In some embodiments, one or more cofactors, coenzymes and additional orancillary proteins may be used to promote or enhance activity of thetest oxidation enzyme, coupling enzyme, or both.

In a preferred embodiment, it is not necessary to recover test enzymefrom host cells that express them, because the host cells are used inthe screening method, in a so-called “whole cell” assay. In thisembodiment, substrate, oxygen donor, and other components of thescreening assay, are supplied to the transformed host cells or to thegrowth media or support for the cells. In one form of this approach, thetest enzyme is expressed and retained inside the host cell, and thesubstrate, oxygen donor, and other components are added to the solutionor plate containing the cells and cross the cell membrane and enter thecell. Alternatively, the host cells can be lysed so that allintracellular components, including any recombinantly expressedintracellular enzyme variant, can be in direct contact with any addedsubstrate, oxygen donor, and other components.

Resulting oxygenated products are detected by suitable means. Forexample, the oxidation product may be a colored, luminescent, orfluorescent compound, so that transformed host cells that produce moreactive oxidation enzymes “light up” in the assay and can be readilyidentified, and can be distinguished or separated from cells which donot “light up” as much and which produce inactive enzymes, less activeenzymes, or no enzymes. A fluorescent reaction product can be achieved,for example, by using a coupling enzyme, such as laccase or horseradishperoxidase, which forms fluorescent polymers from the oxidation product.A chemiluminescent agent, such as luminol, can also be used to enhancethe detectability of the luminescent reaction product, such as thefluorescent polymers. Detectable reaction products also include colorchanges, such as colored materials that absorb measurable visible or UVlight.

To improve the activity of P450 BM-3 or other cytochrome P450 enzymestowards alkanes by directed evolution, a rapid, reproducible screen thatis sensitive to small changes (<2-fold) in activity is desirable(Arnold, 1998). Therefore, an alkane analog such as 8-pnpane (see FIG. 2and Example 1), can be prepared that generates yellow color uponhydroxylation. This “surrogate” substrate with a C8 backbone and ap-nitrophenyl moiety is an analog of octane, and allows use of acolorimetric assay to conveniently screen large numbers of P450 BM-3 orother cytochrome P450 variants mutants for increased hydroxylationactivity in microliter plates (Schwaneberg et al., 1999; Schwaneberg etal., 2001). Hydroxylation of 8-pnpane generates an unstable hemiacetalwhich dissociates to form (yellow) p-nitrophenolate and thecorresponding aldehyde (FIG. 2). The hydroxylation kinetics of hundredsof mutants can then be monitored simultaneously in the wells of amicrotiter plate using a plate reader (FIG. 3) (Schwaneberg et al.,2001). This method is particularly suitable for detecting P450 variantwith improved alkane-oxidation activity.

To screen for improved solvent-resistance, in particular for P450 BM-3variants, a substrate such as 12-pNCA can be added together with anorganic co-solvent (e.g., tetrahydrofurane (THF), DMSO, ethanol,methanol, acetone, etc.) and 12-pNCA conversion initiated by adding aIsocitric co-factor regeneration solution (e.g., Isocitric acid 20 mM;dH₂O, NADP+ 3 mM, Isocitric dehydrogenase 0.8 U/ml). After visible colordevelopment, the reaction can be stopped by adding UT-buster (NaOH 1.5M, 1.5 M Urea, 50% (v/v) DMSO), and absorption at 410 nm recorded.

Enzyme variants displaying improved levels of the desired activity orproperty in the screening assay(s) can then be expressed in higheramounts, retrieved, optionally purified, and further tested for theactivity or property of interest.

Activity Assays

The cytochrome P450 variants created by directed evolution and selectedfor a desired property or activity can be further evaluated by anysuitable test or tests known in the art to be useful to assess theproperty or activity. For example, the enzyme variants can be evaluatedfor their alkane-oxidation capability, alkene-oxidation capability,and/or organic-solvent resistance.

An assay for alkane-oxidation capability essentially comprisescontacting the cytochrome P450 variant with a specific amount of alkanesubstrate, or a substrate which is an alkane analog such as 8-pnpane, inthe presence of an oxygen donor, and any other components (e.g., NADPH)that are necessary or desirable to include in the reaction mixture, suchas NADPH and buffering agents. After a sufficient incubation time, theamount of oxidation product formed, or, alternatively, the amount ofintact non-oxidized substrate remaining, is estimated. For example, theamount of oxidation product and/or substrate could be evaluatedchromatographically, e.g., by mass spectroscopy (MS) coupled tohigh-pressure liquid chromatography (HPLC) or gas chromatography (GC)columns, or spectrophotometrically, by measuring the absorbance ofeither compound at a suitable wavelength. By varying specific parametersin such assays, the Michaelis-Menten constant (K_(m)) and/or maximumcatalytic rate (V_(max)) can be derived for each substrate as is wellknown in the art. Preferred substrates include, but are not limited to,methane, ethane, propane, butane, pentane, hexane, heptane, octane, andcyclohexane. In addition, in particular by HPLC and GC techniques,particularly when coupled to MS, can be used to determine not only theamount of oxidized product, but also the identity of the product. Forexample, octane can be oxidized to octanol where the hydroxyl group ispositioned on any of the carbon atoms in the octanol molecule.

Alkene-oxidation can be evaluated by methods similar to those describedfor alkanes, simply by replacing an alkane with the correspondingalkene, and designing an assay which promotes and detects epoxideformation of the alkene. For example, an assay which detects NADPHconsumption may be used. Preferred alkene substrates include ethene,propene, butene, pentene, hexene, heptene, and octene.

Organic solvent resistance of a cytochrome P450 variant isadvantageously evaluated by conducting an oxidation reaction in thepresence of a certain amount of organic solvent or co-solvent. Thisamount can be varied from, e.g., about 0.1% to about 99.9% (v/v) organicsolvent or co-solvent, more preferably from about 0.5% to about 50%(v/v) organic solvent or co-solvent, and, most preferably, from about 1%to about 10% (v/v) organic solvent or co-solvent, of the total reactionvolume. The amount of oxidation product is then detected as a measure ofthe organic-solvent resistance of the enzyme variant. Such assays can beconducted using various amounts of solvent or co-solvent, and on enzymevariants stored for various periods of time in solutions comprising acertain amount of organic solvent or co-solvent. Preferred organicco-solvents include THF, DMSO, acetone, acetonitrile, and ethanol.

P450 BM-3 Variants

Described herein are several mutations that have been identified toimprove the alkane-oxidation activity and/or alkene-oxidation activity.Thus, a P450 BM-3 variant of the invention can comprise at least one ofthese mutations, optionally in combination with another mutationsselected from the ones described in Table 1A, a mutation not describedin Table 1A, or no other mutation. The variant P450 BM-3 enzymes of theinvention can have a higher oxidation activity towards a saturatedhydrocarbon, e.g., octane, hexane, cyclohexane, propane, ethane, and/orbutane, than wild-type P450 BM-3. Preferred amino acid mutations arethose listed in Table 1A. The skilled artisan could easily identify P450BM-3 variants, including variants comprising truncated, deleted, andinserted amino acid sequences, that comprise one or more of thesemutations and that show enhanced alkane-oxidation activity in a suitableassay as compared to wild-type P450 BM-3.

As identified in Example 3, the particularly active P450 BM-3 mutantsIX139-3 and “J” comprised 11 and 10 amino acid mutations, respectively;V78A, H138Y, T175I, V178I, A184V, H236Q, E252G, R255S, A290V, A295T, andL353V for IX139-3; and V78A, T175I, A184V, F205C, S226R, H236Q, E252G,R255S, A290V, and L353V for “J”. Using wildtype P450 BM-3 as the parent,a clone with approximately 2-fold increased activity for 8-pnpane wasisolated. A single base mutation, threonine (T) to glycine (G), wasfound, which resulted in the amino substitution H236Q. Using H236Q asthe parent for the 2nd generation, 9 mutants were identified in the 2ndgeneration with at least a 2-fold increase in activity. Recombining the9 clones from the 2nd generation, a single clone was identified with 3amino acid substitutions at V78A, H236Q, and E252G. These mutations,were noted as particularly effective mutations for improvingalkane-oxidation activity, since these mutations were preservedthroughout the evolutionary process. For example, H236Q was found in thefirst generation of mutants, and was preserved throughout the process,and the mutations V87A and E252G were discovered in the recombination of9 clones from the 2^(nd) generation mutants, and thereafter preserved.Accordingly, a P450 BM-3 mutant comprising at least one, preferably atleast two, and most preferably all three of these mutations, or anucleic acid encoding such mutants, is a preferred embodiment of theinvention.

In addition, the invention provides P450 BM-3 variants with a higherorganic solvent resistance, especially towards water-miscibleco-solvents such as DMSO and THF. A P450 BM-3 variant with improvedorganic solvent-resistance according to the invention comprises at leastone of the mutations in Table 1A, optionally in combination with othermutations selected from the ones described in Table 1A, a mutation notdescribed in Table 1A, or no other mutation. As noted in Example 2, P450BM-3 variants having particularly improved solvent resistance (F87A5F5and W5F5) comprised R471A, E494K, and S1024E mutations, optionally withthe mutation F87A. Accordingly, a P450 BM-3 mutant comprising at leastone, preferably at least two, and most preferably all three of thesemutations, with or without the F87A mutation, or a nucleic acid encodingsuch mutants, is a preferred embodiment of the invention. A skilledartisan could easily identify P450 BM-3 variants, including variantscomprising truncated, deleted, and inserted amino acid sequences, thatcomprise one or more of the mutations in Table 1A and that show improvedorganic solvent-resistance in a suitable assay as compared to wild-typeP450 BM-3.

Directed evolution techniques can thus significantly improve the organicsolvent resistance of P450 BM-3 and its mutant P450 BM-3 F87A towardDMSO and THF, or any other organic solvents or co-solvents. It was alsoidentified herein that position F87—located at the end of the substrateaccess channel directly above the heme—plays a size-dependent key rolein modulating the monooxygenase activity in organic co-solvents. Becausethe mutations are located at the interface between the reductase domainand the P450 domain, it is believed, while not being limited to anytheory, that the mutations create tighter domain bonding, which supportelectron transfer from the reductase to the heme in the presence ofcosolvent.

Notably, except for mutation 1024 (which is not in the crystallizedreductase fragment) all of the mutations improving organic solventresistance are located at the interface between heme domain andreducase. It is therefore possible that the mutations “stabilize” theorientation of heme to reducase allowing an electron transfer from thereducase to the heme. Potentially, and without being bound to anytheory, this electron-transfer could be essential, or at leastimportant, for activity.

Preferably, the P450 BM-3 variants of the invention have an at leasttwo-fold improvement in the capability to oxidize a chosen alkane (e.g.,octane, hexane, pentane, butane, cyclohexane, or propane), and/or an atleast two-fold improvement in the organic solvent-resistance, ascompared to wild-type P450 BM-3. Even more preferably, the improvementfor either or both of these properties is at least 3-fold, at least4-fold, at least 5-fold, at least 10-fold, or at least 15-fold.

The P450 mutations of the present invention are wholly unexpected. Forexample, Schmid and coworkers recently described the engineering of P450BM-3 to hydroxylate short chain (C8-C10) fatty acids not accepted by thewildtype enzyme (Li et al., 2001). Guided by the x-ray crystalstructure, they focused on eight individual amino acids within thebinding pocket to perform saturation mutagenesis. This approachsuccessfully discovered mutations (Val26Thr, Arg47Phe, Ala74Gly,Leu188Lys, Phe87Ala) that altered the enzyme's substrate specificity. Anindole-hydroxylating triple mutant of BM-3 (Phe87Val, Leu188G1n,Ala74Gly) was also shown to hydroxylate octane (Appel et al., 2001).Random mutagenesis, however, can discovery other, often subtle ways tomodify activity. For example, sequencing the gene for the P450 BM-3variants (Table 1) revealed that none of the more active mutantsdescribed herein contained any of the mutations found by Schmid andcoworkers. Furthermore, the best mutants described here wereconsiderably more active on octane than the triple mutant reported bySchmid and coworkers (Appel et al., 2001).

Attempts to engineer catalyst specificity are often limited to alteringamino acids directly involved in substrate recognition and binding. Onlyone residue that is in direct contact with substrate in the wildtypeenzyme has been mutated in IX139-3 (V78A). In fact, many of themutations discovered herein were not found in the substrate bindingchannel, as suggested by the structure of P450 BM-3 with the bound fattyacid substrate. Some are in the hydrophobic core of the protein.Furthermore, several of the mutations are found in the F and G helicesand the loop connecting them. This region undergoes the largestmovements upon substrate binding (Li and Poulos, 1997). Mutations inthis region may be responsible for new binding characteristics andactivity for alkanes. How these particular substitutions enhanceactivity towards alkanes is not clear. They would have been verydifficult to identify using currently available structure-based designmethods. The cytotoxicity of the new variants indicates that enzymesshowing such high alkane hydroxylation activity may not be produced byliving cells under natural conditions and could only be generated bylaboratory evolution strategies.

Non-P450 BM-3 Variants

As described above, the invention also provides for novel non-P450 BM-3cytochrome P450 oxygenases in which one or more of the amino acidresidues listed in Table 1A have been conserved. Conservation of anamino acid residue can show that the residue has an important functionfor the oxygenase activity and/or stability of the P450 enzyme. Thus,the P450 BM-3 mutations identified herein to improve alkane-oxidationactivity and/or solvent resistance can simply be translated onto suchnon-P450 BM-3 enzymes to yield improved properties according to theinvention.

Any method can be used to “translate” the P450 BM-3 mutation ontoanother cytochrome P450 enzyme, and such methods are well known in theart. For example, sequence alignment software such as SIM (alignment oftwo protein sequences), LALIGN (finds multiple matching subsegments intwo sequences), Dotlet (a Java applet for sequence comparisons using thedot matrix method); CLUSTALW (available via the World Wide Web asfreeware), ALIGN (at Genestream (IGH)), DIALIGN (multiple sequencealignment based on segment-to-segment comparison, at University ofBielefeld, Germany), Match-Box (at University of Namur, Belgium), MSA(at Washington University), Multalin (at INRA or at PBIL), MUSCA(multiple sequence alignment using pattern discovery, at IBM), and AMAS(Analyse Multiply Aligned Sequences). A person of skill can choosesuitable settings, or simply use standard default settings, in theseprograms to align P450 BM-3 with another cytochrome P450 enzyme. SeeFIG. 20 for representative sequence alignments, and Table 2 forrepresentative non-P450 BM-3 mutations.

Alternatively, such sequence alignments of P450 BM-3 with othercytochrome P450 enzymes can be taken from the literature, and amino acidresidues corresponding to the mutated amino acid residues of theinvention identified. For example, such information can be derived fromde Montellano (1995) (see, especially, FIG. 1 on page 187).

While some P450 enzymes may not share significant sequence similarities,particular domains such as the heme-containing domains of P450s dodisplay close structural similarity (Miles et al., 2000). Therefore, thepositions of the various mutations described here could be translated tosimilar positions in different P450s having very low sequence similarityto P450 BM-3 using molecular modeling of those P450s based on sequencehomology. Examples of using such techniques to model various P450s basedon sequence homology with P450 BM-3 are available (Lewis et al., 1999).The same mutations described here, when placed in their correspondingpositions in other P450 structures (as determined by modeling) wouldconfer similar improvements in alkane-/alkene oxidation activity andorganic solvent resistance.

In this regard, FIG. 21 shows a topological view of a cytochrome P450enzyme, including the various domains of cytochrome P450 enzymes and themutations contemplated by the present invention in each of thosedomains. While the topological view presented in FIG. 21 is that ofP450_(Bm-P), with only minor modifications, this topology diagram may beused for other P450s. Briefly, FIG. 10 shows where mutations disclosedherein were made and these are summarized in Table 3 below.

TABLE 3 Locations of Selected P450 BM-3 Mutations Domain Amino AcidResidue Heme domain: helix B′ V78 Heme domain: loop connecting helicesB′ and C F87 Heme domain: β3-1 H138 Heme domain: helix F T175 Hemedomain: helix F V178 Heme domain: helix F A184 Heme domain: helix F N186Heme domain: helix G D217 Heme domain: helix G S226 Heme domain: helix HH236 Heme domain: helix I E252 Heme domain: helix I R255 Heme domain:helix J A290 Heme domain: helix J A295 Heme domain: β1-3 L353 Hemedomain: loop connecting helices K′ and L G396 — R471 Helix in flavindomain E494 — S1024

Therefore, based on the topological view presented in FIG. 10, a P450variant may be prepared by making one or more mutations in one or moreof the domains of P450 identified in Table 3 above. Further, thetopological view of FIG. 10 allows one to compare BM-3 variants withother P450 enzymes and identify those residues of non-BM-3 enzymes thatcould be mutated according to the secondary and tertiary structuralmotifs within the enzyme(s).

Thus, the invention provides novel, non-P450 BM-3 cytochrome P450oxygenases in which one or more of the amino acid residues listed inTable 1A have been conserved. Conservation of an amino acid residue canshow that the residue has an important function for the oxygenaseactivity and/or stability of the P450 enzyme. The P450 BM-3 mutationsidentified herein to improve utilization of hydrogen peroxide as oxygensource and/or thermostability can simply be translated onto suchnon-P450 BM-3 enzymes to yield improved properties according to theinvention.

Once the corresponding amino acid residues have been identified, aperson of skill can test various mutations of these amino acid residuesto identify those that yield improved alkane-oxidation capability orimproved organic solvent-resistance as compared to the cytochrome P450wild-type enzyme. Preferred amino acid substitutions are those thatcorrespond to a substitution listed in Table 1A for P450 BM-3 mutations.

EXAMPLES

The invention is illustrated in the following examples, which areprovided by way of illustration and are not intended to be limiting.

Example 1 Directed Evolution of a Cytochrome P450 Monooxygenase forAlkane Oxidation

This Example describes the discovery of P450 BM-3 variants. P450 BM-3variants were created and identified by directed evolution techniques,specifically iterative cycles of random mutagenesis and recombination,and functional screening.

All chemical reagents were procured from Aldrich, Sigma, or Fluka.Enzymes were purchased from New England Biolabs, Stratagene, andBoehringer Mannheim. The 1H NMR spectrum was recorded on a Varian 300MHz nuclear magnetic resonance spectrometer with a Mercury console.Quantitative Technologies Inc. (Whitehouse, N.J.) performed elementalanalysis.

A. Random Library Generation and Screening of P450 BM-3

P450 BM-3 modified to contain a His6 tag was amplified from pT-USC1 BM-3(Schwaneberg et al., Anal Biochem 1999a, 269:359-366) by PCR techniquesusing a proofreading polymerase Pfu to introduce a BamHI upstream of thestart codon and an EcoRI site immediately downstream from the stopcodon. The two oligonucleotides used were as follows:

(BamHI site underlined, SEQ ID NO: 7)5′-CGCGGATCCATCGATGCTTAGGAGGTCATATGACAATTAAAGAAATG CCTC-3′(EcoRI site underline, SEQ ID NO: 8)5′-CCGGAATTCTTAATGATGATGATGATGATGCCCAGCCCACACGTCTT TTGC-3′

The PCR product was digested with BamHI and EcoRI. The P450 BM-3 genewas ligated into expression vector pCWOri (+) (Barnes, 1996) (p BM-3WT18-6), which is under the control of double Ptac promoter and containsan ampicillin resistance coding region. A silent mutation was introducedto construct a SacI site 130 bases upstream of the end of the hemedomain. The QuikChange (Stratagene) protocol was followed and theprimers were as follows:

(SacI site underlined; SEQ ID NO: 9)5′-CATACAAACTACGAGCTCGATATTAAAGAAAC-3′(SacI site underlined; SEQ ID NO: 10)5′-GTTTCTTTAATATCGAGCTCGTAGTTTGTATG-3′

Synthesis of p-nitrophenoxyoctane (8 pnpane)

1-bromooctane (1 g, 5.18 mmole) and 4-nitrophenol, sodium salt (0.92 g,5.71 mmole) were refluxed in DMSO (30 ml) at 120° C. for 5 hours. TheDMSO was distilled off to near dryness. The resulting brown residue wasloaded onto a silica column and eluted with 10:1 mixture of petroleumether and diethylether. The yield was 30%. 1H NMR (CDCl₃, peaks at =8.18(m, 2H), 6.93 (m, 2H), 4.04 (t, 2H), 1.81 (p, 2H), 1.33 (m, 10H), 0.89(t, 3H)). Elemental analysis calculated for C₁₄H₂₁O₃N: C, 66.91; H,8.42; N, 5.57. Found: C, 66.97; H, 8.34; N, 5.52.

Expression and Purification of P450 BM-3 Variants

The P450 BM-3 gene, which includes a silent mutation to introduce a SacIsite 130 bp upstream of the end of the heme domain, was cloned behindthe double tac promoter of the expression vector pCWori (p BM-3_WT18 6)(Farinas et al., 2001). This plasmid was used for production of wildtypeprotein and as a starting clone for directed evolution. For proteinproduction, Terrific Broth (TB) media (500 ml) supplemented with traceelements (125 μL: 0.5 g MgCl₂, 30.0 g FeCl₂ 6H₂O, 1.0 g ZnCl₂ 4H₂O, 0.2g CoCl₂ 6H₂O, 1.0 g Na₂MoO₄ 2H₂O, 0.5 g CaCl₂ 2H₂O, 1.0 g CuCl₂ and 0.2g H₂BO₃ in 1 L HCl solution (90% v/v distilled water: concentrated HCl))(Joe et al., 1999) was inoculated with 500 μl of an overnight culture ofE. coli BL21 containing the expression plasmid. After shaking for 10hours at 35° C. and 250 rpm, aminolevulinic acid hydrochloride (ALA)(0.5 mM) was added, and expression was induced by addition of IPTG (1mM) and cells were harvested by centrifugation after a total cultivationtime of 30 hours.

After 30 hours, the cells were harvested by centrifugation and thesupernatants discarded. The pellets were washed with Tris HCl (15 ml, pH8.3). Cells were resuspended in Tris HCl (15 ml, pH 8.3), sonicated(2×45 sec; output control=7, duty cycle 40%; Sonicator, Heat SystemsUltrasonic, Inc.) and centrifuged. The supernatants were further clearedthrough a 0.45 μM filter. The filtrate was diluted 15 mL water andpurified by published procedures (Farinas 2001). P450 BM-3concentrations were measured from the CO difference spectra (Omura1963).

Library Construction

For the first 2 generations, mutagenic PCR was performed on the hemedomain in a 100 mL reaction as described in Zhao et al. (1999) with somemodifications. The mutated P450 BM 3 fragment was 1291 base pairs. Thereaction contained MgCl₂ (7 mM) and the following forward and reverseprimers (40 pmol each):

(Forward, SEQ ID NO: 11) 5′ ACAGGATCCATCGATGCTTAGGAGGTCATATG 3′(Reverse, SEQ ID NO: 12) 5′ GTGAAGGAATACCGCCAAG 3′.

The reaction also contained p BM-3 WT18-6 (10 ng), dNTPs (0.2 mM dGTP,0.2 mM dATP, 1 mM dCTP, 1 mM dTTP), and Taq polymerase (5 units, Roche),KCl (50 mM), and Tris-HCl (10 mM, pH 8.3, 20° C.). MnCl2 (0.0, 0.05, and0.1 mM) was added to the PCR mixture to alter the error rate of thepolymerase. PCR was performed in a thermocycler (PTC200, MJ Research,Waltham, Mass.) for 30 cycles (95° C., 45 s; 50° C., 30 s; 72° C., 2min). The PCR product was restricted with BamHI and SacI and ligatedinto expression vector pCWOri (+). The resulting plasmid was transformedinto E. coli strain DH5α and the colonies were selected on agar platescontaining ampicillin (100 mg/ml).

For the 3^(rd) generation: 9 mutants from the 2^(nd) generation showingat least 2-fold improved activity on 8-pnpane were recombined bystaggered extension process (StEP) (Zhao H M, et al. Nature Biotechnol1998; 16:258 261). Recombination was done in 50 μL reactions asdescribed (Zhao et al., 1999). Each reaction contained buffer (Qiagen1×PCR buffer), template (10 ng each), forward and reverse primer (final0.15 mM), dNTPs (200 μM each) and Taq DNA polymerase (2.5 units,Qiagen). PCR was performed in a thermocycler (PTC200, MJ Research,Waltham, Mass.) (1. 95° C., 2 min; 2. 95° C. 30 s; 3. 50° C., 10 s;repeat steps 2 and 3 100×). The PCR product was restricted with BamHIand SacI and ligated into expression vector pCWOri (+). The resultingplasmid was transformed into E. coli strain DH5a and the colonies wereselected on agar plates containing ampicillin (100 mg/ml).

For the 4^(th) and 5^(th) generation, error prone PCR was also performedusing the GeneMorph PCR Mutagenesis Kit (Stratagene) applying conditionsof high error rate (1-10 ng template DNA). The PCR product wasrestricted with BamHI and SacI and ligated into expression vector pCWOri(+). The resulting plasmid was transformed into E. coli strain DH5a andthe colonies were selected on agar plates containing ampicillin (100mg/ml).

Recombination was done in 50 mL reactions as described (Zhao et al.,1999). Each reaction contained buffer (Qiagen 1×PCR buffer), template(10 ng each), forward and reverse primer (final 0.15 mM), dNTPs (200 μMeach) and Taq DNA polymerase (2.5 units, Qiagen). PCR was performed in athermocycler (PTC200, MJ Research, Waltham, Mass.) (1. 95° C., 2 min;2.95° C. 30 s; 3. 50° C., 10 s; repeat steps 2 and 3 100×).

Screening for Hydroxylation Activity Cultivation and Expression of P450BM-3 Mutant Libraries

For the first three rounds of evolution, a robot (Qpix, Genetix) pickedand inoculated colonies into 1 ml deep-well plates containing LB media(400 ml) and ampicillin (100 mg/ml). The plates were incubated at 37°C., 270 RPM, and 80% relative humidity. After 24 hours, the cultureliquid (50 ml) was added to TB (450 ml) containing, ampicillin (100 mgampicillin/ml), thiamine (5 mg/ml), and trace elements (0.25 ml/ml).After growth at 37° C. for 1 hour, aminolevulinic acid hydrochloride (1mM) and isopropyl - - - thiogalactopyranoside (1 mM) was added. Thetemperature was shifted to 30° C. and the cultures were grown for 24hours.

For the 4^(th) and 5^(th) generations the screening procedure wasmodified to the following procedure. The plates with the picked colonieswere incubated in LB (containing 100 μg/L ampicillin) at 30° C., 270RPM, and 80% relative humidity. After 24 hours, TB (500 ml) containing,ampicillin (100 mg ampicillin/ml), thiamine (5 mg/ml), and traceelements (0.25 ml/ml), δ-aminolevulinic acid hydrochloride (1 mM) and 10μM isopropyl-thiogalactopyranoside, was inoculated with the precultureusing a 96 inoculation pin and grown for 24 hours.

Preparation of Cell Lysates

For the first three generations, the plates were centrifuged andsupernatants were discarded. Cell pellets were washed with Tris-HCl (350ml, pH 8.3), frozen at −20° C. for at least 8 hours and then resuspendedin 400 μl Tris-HCl (350 ml, pH 8.3) containing lysozyme (0.5 mg/ml),deoxyribonuclease I (0.1 mg/ml) and MgCl₂ (10 mM). After incubation at37° C. for 45 minutes, the plates were centrifuged and the lysate (150ml) was transferred to a 96-well plate. For the final generations, thefrozen cell pellets were resuspended in phosphate buffer (1 mL, 0.1 M,pH 8.0) containing lysozyme (0.5 mg/mL0, DnaseI (0.1 μL/mL) and MgCl₂(10 mM). The lysates were centrifuged and the supernatants were dilutedfor activity measurements in 96 well microtiter plates.

High Throughput Determination of Enzymatic Activity

For the first three generations, 8-pnpane (150 μM) in DMSO (1%) wasadded to the lysate and incubated at room temperature. After 5 minutes,NADPH (1 mM) was added and the absorbance at 410 nm was measured with amicroplate spectrophotometer (SPECTRAmax, Molecular Devices).

For the final generations, mutant libraries were screened using 8-pnpaneas described above. Also, a cofactor (NADPH) depletion assay was used todetermine the turnover rates. The lysates were diluted into 96 wellmicrotiter plates containing phosphate buffer (200 μL, 0.1 M, pH 8.0),alkane substrate (0.5-1.0 mM), and DMSO (1%). The liquid alkanes wereadded to the buffer using alkane stock solutions in DMSO, whereasgaseous alkanes were bubbled into buffer for ˜45 minutes to obtainsaturated solutions. The reaction was initiated by addition of NADPH(200 μM), and the oxidation of NADPH was monitored at 340 nm. Only themutants active in both screens were isolated and recharacterized.

Rescreening

The most active clones from the primary screen were streaked out on agarplates to get single colonies. Four to 8 single colonies were reculturedin deep well plates and rescreened as described above. In the rescreenall clones were also assayed for hydroxylation of the target substrateoctane. The same dilutions of lysates in buffer were used as in the8-pnpane assay. A stock octane substrate solution (225 μM) in DMSO (1%)was added to the lysates. After addition of NADPH (0.75 mM), in the samebuffer as used for the lysates, the oxidation of NADPH to NADP⁺ wasfollowed using the microplate spectrophotometer for 3 min at 340 nm. Asa control the same assays were performed without addition of the alkanesubstrate to verify that NADPH consumption was coupled to the presenceof substrate.

For propane oxidation, the cells were already lysed in a buffer, wherethe 0.1 M Tris-HCl from the lysis buffer described above was replaced by30 mM phosphate buffer pH 7.4 to avoid organic substances (which couldconceivably become substrates for the enzyme) in the buffer. Phosphatebuffer was saturated with propane by bubbling propane into the bufferfor 1 hour. Thirty μl of the bacterial lysates were pipeted into 96 wellmicrotiter plates and 120 μl of the propane saturated buffer were added.The reaction was started again by addition of 50 μl of 3 mM NADPH andfollowed at 340 nm for 3 mM.

Agar Plate Colony Development

Cells were grown on LB agar plates containing 100 μg/ml ampicillin, 1 mMaminolevulinic acid hydrochloride and 10 μM isopropyl.thiogalactopyranoside. The latter two substances are not necessary ifthe activity resulting from a leaky promoter system is high enough. Asubstrate solution was prepared containing 30 mM phosphate buffer pH7.4, 100 μM polymyxin B sulfate as a cell permeabilizer, 2 mM NADPH and5 mM alkane substrate. The substrate solution was sonicated before useto emulsify most of the substrate in the buffer system.

A nitrocellulose membrane was soaked with this substrate solution andplaced on the colonies on the agar plate. The optimal reaction time wasestimated by testing the assay on control agar plates with colonies ofclones with different hydroxylation activity before starting the mainscreen. After the reaction time the color reagent (0.5 mg/ml NBT in 30mM phosphate buffer pH 7.4 and some crystals of the catalyst PMS) waspipeted directly onto the nitrocellulose membrane. Colonies locatedunder white spots on the membrane were picked with a toothpick andstreaked out on fresh agar plates to get single colonies for there-screen.

Determination of the Maximum Initial Rates for Hydroxylation

The enzymes were purified and quantified as described above. First, thesubstrate concentration corresponding to the maximum turnover rate wasdetermined by monitoring NADPH consumption with a plate reader in thepresence of enzyme in phosphate buffer (0.1 M pH 8.0) and varyingamounts of substrate in methanol (1%). After identifying theconcentration of substrate that coincides with the maximum rate, therate was measured using an UV-Vis spectrophotometer and 1 cm path lengthquartz cuvettes. A typical reaction solution contained enzyme (700 μL,0.35-3.5 μM) in potassium phosphate buffer (0.1 M, pH 8.0) and substratein methanol (1%). The reaction was initiated by the addition of NADPH(300 μL, 200 μM), and the absorption at 340 nm was monitored.

The amount of H₂O₂ was determined using2,2′-azino-di-[3-ethyl-benzothiazidine-6-sulfonic acid/horseradishperoxidase assay by following published procedures (Yeom, H. & Sligar,S. G. Oxygen activation by cytochrome P450BM-3: effects of mutating anactive site acidic residue. Arch Biochem Biophys 337, 209-216. (1997).

B. Wild-Type P450 BM-3 Alkane Oxidation Assay Octane Oxidation by P450BM-3

The enzymes were purified and quantified as described above. Theoxidation was performed with solutions containing octane in DMSO (1 mMoctane; 1% DMSO), P450 BM-3 (2-3 μM), and NADPH (1-5 mM) in Tris-HCl (50mM) containing NaCl (340 mM). A concentrated solution of octane in DMSOwas added to the enzyme. The resulting solution was incubated for 30minutes at room temperature. Octane oxidation was initiated by theaddition of NADPH in aqueous solution. After a specific time, thesolution was extracted three times with of CH₂Cl₂ (333 mL) containingdecanol (1 mM) as an internal standard. The organic layer was dried overanhydrous Na2SO4, and the products were analyzed by GC/MS.

Catalytic activity of P450 BM-3 was measured spectrophotometrically bymonitoring the rate of NADPH oxidation, as described (Matson et al.(1977). The assay solution contained 0.1 nmole P450 BM-3, octane inDMSO, and 0.8 mM NADPH, 50 mM NaCl in 0.1 M Tris-HCl, pH 8.2.

Production of H₂O₂ during the hydroxylation reaction was determinedusing the iron(III) thiocyanate assay (Fruetel et al., 1994). 1.0×10⁻⁹mole P450 BM-3 was incubated with 1.0×10⁻⁶ mole octane for 5 minutes.The reaction was initiated by addition of 2.5×10⁻⁷ mole NADPH to theenzyme solution. Every 2 minutes a 0.2 ml aliquot from the reaction wasadded to 1.0 ml aqueous iron(II) solution (5.0 grams FeSO₄(NH₄)₂×6H₂O,45.0 ml degassed H₂O, 5 ml concentrated H₂SO₄). Subsequently, 0.4 ml ofa 10% aqueous solution of KSCN was added to the solution, and theabsorbance was measured at 480 nm.

C. Assay for IX139-3 Catalytic Activity with Alkanes Determination ofthe Maximum Initial Rates for Liquid Alkane Hydroxylation by IX139-3

The enzymes were purified and quantified as described above. First, thesubstrate concentration corresponding to the maximum turnover rate wasdetermined by monitoring NADPH consumption with a plate reader in thepresence of enzyme in phosphate buffer (0.1 M pH 8.0) and varyingamounts of substrate in methanol (1%). After identifying theconcentration of substrate that coincides with the maximum rate, therate was measured using an UV-Vis spectrophotometer and 1 cm path lengthquartz cuvettes. A typical reaction solution contained enzyme (700 μL,0.35-3.5 μM) in potassium phosphate buffer (0.1 M, pH 8.0) and substratein methanol (1%). The reaction was initiated by the addition of NADPH(300 μL, 200 μM), and the absorption at 340 nm was monitored. Thesubstrates examined were pentane, hexane, cyclohexane, and octane.

The amount of H₂O₂ was determined using2,2′-azino-di-[3-ethyl-benzothiazidine-6-sulfonic acid/horseradishperoxidase assay by following published procedures (Yeom & Sligar,1997).

Determination of the maximum initial rate for gaseous alkanehydroxylation by IX IX139-3

Potassium phosphate buffer (0.1 M, pH 8.0) was saturated with thegaseous alkane (propane or butane) by bubbling the substrate into thesolution for 1 hour. A typical reaction contained alkane saturatedbuffer (700 μL) and enzyme (0.5 μM). Addition of NADPH (300 μL, 200 μM)in buffer initiated the reaction, and the rate of NADPH oxidation wasfollow at 340 nm.

GC/MS Analysis

Biocatalytic oxidations were performed under oxygen limited conditionsin sealed vials. For octane, hexane, or cyclohexane conversions, thesolution contained alkane (1 mM) in DMSO (1% DMSO) and enzyme (1 μM) inpotassium phosphate buffer (100 mM, pH 8.0). The solution was stirred atroom temperature for 5 minutes, and the reaction was initiated by theaddition of NADPH (1 mM).

Reactions with gaseous alkanes were carried out in a sealed 20 mL vialcontaining enzyme (1.0 μM) in potassium phosphate buffer (5 mL, 100 mM,pH 8.0). The headspace was filled with either propane or butane. Thereaction was initiated with the addition of NADPH (1 mM). The reactionmixture was analyzed directly by GC/MS using an Hewlett Packard 5890Series II gas chromatograph coupled with an Hewlett Packard 5972 SeriesMass Selective Detector. The GC was fitted with HP FFAP column(crosslinked FFAP, 30m×0.25 mm×0.25 mm film thickness). The conditionfor octane is as follows: Isothermic at 120° C. for 6 minutes. Thecondition for hexane and cyclohexane: (1) 100° C. for 5 minutes to 50°C. (2) 100° C. to 200° C. at 25° C./min. (3) Isothermic at 200° C. for 2minutes. The condition for propane and butane: (1) 30° C. for 3 minutes.(2) 30° C. to 150° C. at 20° C./min. Authentic standards were used toidentify the products by retention time. Products were further verifiedby matching the fragmentation distributions with a database in thesoftware provided with the instrument manufacturer.

Initially for octane oxidation by wildtype, the products were identifiedwith GC/MS using a Hewlett Packard 5890 Series II gas chromatographcoupled with a Hewlett Packard 5989A mass spectrometer. The GC wasfitted with an HP 1 column (crosslinked methyl silicone gum, 12 m×0.2mm×0.33 mm). The temperature gradient is as follows: 1) 40 to 50° C. at15° C., 2) 50 to 75° C. at 10° C./min, 3) 75 to 160° C./min. Authenticstandards were used to identify the retention times of the products. Theproducts were further verified by matching the fragmentationdistributions with a database in the software provided by the instrumentmanufacturer.

D. Results Wildtype P450 BM-3 is Active Towards Octane

In the presence of purified P450 BM-3 and NADPH, octane was consumedwithin 2 hr and gave several products detectable by GUMS (FIG. 1). Thetotal yield of product detected was about 50%, with alcohols accountingfor about 90% of the product and ketones representing 10%. The majorhydroxylated products were 4-octanol, 3-octanol, and 2-octanol.1-Octanol was not detected under the experimental conditions applied.The product ratio was, approximately, 8:9:1; 4-octanol: 3-octanol:2-octanol. 4-Octanone and 3-octanone were also present in the productmixture. A possible mechanism for the formation of the ketones ishydroxylation of the alcohol to generate a gem-diol which dehydrates tothe corresponding ketone (Boddupalli et al., 1992; March, 1992a).Another possible mechanism is via the pinacol rearrangement (March,1992b). However, it is also possible that traces of protein impuritieswere responsible for this oxidation, and further tests were thereforeperformed to validate the results.

It was tested whether P450 BM-3 catalyzes the oxidation of 3-octanol to3-octanone. Upon addition of NADPH to a solution of P450 BM-3 and3-octanol, a peak at 3.7 min appeared. This peak had the same retentiontime as an authentic sample of 3-octanone. Furthermore, thefragmentation pattern of the peak matched that of 3-octanone found inthe mass spectrum database. Solutions containing only P450 BM-3 and3-octanol, but no NADPH, do not produce any detectable 3-octanone.Similar results were obtained with 4-octanol as the substrate.

The activity of P450 BM-3 towards octane was measuredspectrophotometrically by monitoring the rate of NADPH consumption. Thefollowing results were obtained: k_(cat)=0.7 s⁻¹, K_(m)=2.0×10⁻⁵ M andk_(cat)/K_(m)=3.5×10⁴ M⁻¹s⁻¹. Thus, on octane, the P450 BM-3 enzyme hasa kcat 120-fold less and a Km that is 15 times larger(k_(cat)/K_(m)˜2000 times less) than on its preferred C16 fatty acidsubstrate.

P450 BM-3 Catalyzes Hydroxylation of Octane without Uncoupling

Hydrogen peroxide production during catalysis by P450 BM-3 was alsomonitored in order to determine whether the rate of NADPH oxidation wasaffected by uncoupling (Oliver et al., 1997; Fruetel et al., 1994). H₂O₂was not detected under the experimental conditions, which indicates thatthe P450 catalyzes the hydroxylation of octane without significantuncoupling. This was consistent with previous reports that indicatedefficient coupling for P450 BM-3 acting on unnatural substrates such asstyrene or alkyl trimethylammonium compounds (Oliver et al., 1997;Fruetel et al., 1994).

Directed Evolution Improves the Activity of Cytochrome P450 BM-3 TowardsOctane and Other Substrates

Mutant library construction was focused on the P450 BM-3 heme domain,which contains the substrate binding site and the monooxygenaseactivity. The libraries were generated by error prone PCR (Zhao et al.,1999), using MnCl₂ concentrations of 0.0, 0.05, and 0.1 mM, whichyielded 70, 60, and 50% active transformants, respectively. The librarygenerated with no added MnCl₂, corresponding to about 2 base changes pergene (0.15% error rate), was chosen for screening (Zhao et al., 1999).

Two thousand clones of the first generation library were screened and 23of the most active clones were selected for further analysis. Since thescreen was sensitive to total activity, which includes increasedexpression as well as changes in specific activity, enzymeconcentrations were estimated from the CO difference spectra for bindingto the reduced P450 BM-3 in order to calculate specific activities(Omura and Sato, 1964). The best variant, VIII118 2C9, displayedapproximately twice the specific activity of the wildtype enzyme (FIG.4).

The 2^(nd) generation library was also created with error-prone PCRusing VIII118 2C9 as the template, and 9 mutants were isolated that werebetween 1.8 and 2.5 times more active than VIII118 2C9. 1×18_(—)18 wasfound to be 2.38 times more active than VIII118 2C9, and it was used forcomparison for the 3^(rd) generation library.

The 3^(rd) generation was produced by StEP recombination (Zhao et al.,1999), using 9 variants from the 2^(nd) generation, and the most activemutant (IX35_(—)4) was found to be 1.74 times more active thanIX118_(—)18. IX35_(—)4 was used for comparison for the 4^(th)generation.

The 4^(th) generation was constructed by error-prone PCR using theIX35_(—)4 as the template. The GeneMorph PCR Mutagenesis Kit(Stratagene) was used to create the libraries. The most active variant(IX 79_(—)1) was 1.35 times as active compared to IX35_(—)4. IX79_(—)1was used for comparison for the 5^(th) generation library.

After some generations the evolved P450 BM-3 variants began showing highcytotoxicity. This caused large differences between the activitiesmeasured in the high throughput screen and then again afternormalization to CO binding. As a result, the improvements in activitybecame smaller in generation 4, which might indicate that there is a“plateau”, i.e., a rather low limit to the activity which is possiblefor a P450 BM-3 variant created by this approach alone.

Even in the case of the strongly regulated promoter used in this study,there is always some leakiness of the promoter. Even small amounts oftoxic proteins produced by the leaky promoter could cause large(negative) physiological effects on the different clones during growthin the 96 deep well plates. Therefore, the screening procedure wasredesigned after generation four, as described under “Materials andMethods.”

The 5^(th) generation was produced using 1×79_(—)1 as the template.Again, Genemorph was used to create the libraries. Using a modifiedscreening procedure in generation five resulted in highly improvedvariants for alkane hydroxylation. The best three clones are between1.24-1.86 times more active than IX79_(—)1. The best clone from the5^(th) generation (IX139-3) was 23 times more active than wildtype. Thesame library of the 5^(th) generation was used for a second screen usingmutant IX139-3 for comparison.

Selected clones, including the three best clones from the first screen(IX139-3, 1×139-37 and 1×139-43), were recombined by StEP to produce thelibrary of the 6^(th) generation. Several hundred clones of this newlibrary have been analyzed using the microtiter plate assays, andresults indicate further improvement of the alkane hydroxylatingactivity. For example, the mutant “J” (V78A, T175I, A184V, F205C, S226R,H236Q, E252G, R255S, A290V, and L353V) was shown to be 2.0 and 1.6 foldmore active for NADPH consumption for propane and butane, respectively.

All improved clones found in the primary screen were rescreened forNADPH consumption by hydroxylation of octane. Only clones showingimproved activity in presence of octane and a low background withoutoctane were selected as templates for new generations. This gave somecertainty that this was the right track for optimizing alkanehydroxylation activity. An assay for propane hydroxylation was includedin the screening program in the sixth generation. The mutations, and theamino acid substitutions they lead to in the evolved P450 BM-3 enzymes,are listed below (Table 4).

TABLE 4 Selected Cytochrome P450 Mutants Created By Directed EvolutionMutations were verified by sequencing one strand of DNA. Amino AcidCodon Generation Clone Mutation Mutation Wild-type viii18_6 1st Gen.,viii118 2C9 His236Gln CAT→CAG ep PCR, taq 2nd Gen., viii159 7A4 Met30IleATG→ATT ep PCR, taq Asp232Gly GAT→GGT His236Gln CAT→CAG Met416LeuATG→TTG viii159 7G4 Glu64Ala GAA→GCA Ile220thr ATT→ACT His236Gln CAT→CAGThr411Ala ACG→GCG viii159 10A6 Val78Ala GTA→GCA Phe162Ser TTT→TCTLys224Ile AAA→ATA His236Gln CAT→CAG Lys306Glu AAA→GAA viii159 10A11Thr10? ACG→NCG Glu13? GAG→GNG Met118Leu ATG→TTG Gly154Gly* GGT→GGAHis236Gln CAT→CAG viii159 12A5 His236Gln CAT→CAG Ile258Thr ATT→ACTix18_12 Pro45Pro* CCT→CC(T/C) His171Gln CAT-CAG His236Gln CAT→CAGAsp370G;u GAT→GAA ix18_18 Gln73Gln* CAA→CAG His236Gln CAT→CAG Ile259Va1ATT→GTT Leu272Leu* CTT→CTC Lys289Lys* AAA→AAG Glu380Gly GAA-GGA ix18_34ALys187Glu AAG→GAG His236Gln CAT→CAG ix18_38 Lys59Lys* AAA→AAG Lys97Lys*AAA→AAG His236Gln CAT→CAG Glu252Gly GAG→GGG Lys289Lys* AAA→AAG3rd gen, step ix35_4 Val78Ala GTA→GCA His236Gln CAT→CAG Glu252GlyGAG→GGG 4th gen, ix79_1 Val78A1a GTA→GCA genemorph Phe107Phe* TTC→TTTThr175Ile ACA-ATA Ala184Val GCA-GTA His236Gln CAT→CAG Glu252Gly GAG→GGGAla290Val GCA→GTA Leu353Val CTA→GTA 5th gen, ix139-3 Val78Ala GTA→GCAgenmorph Phe107Phe* TTC→TTT His138Tyr CAT→TAT Thr175Ile ACA→ATAVal178Ile GTC→ATC Ala184Val GCA-GTA His236Gln CAT→CAG Glu252Gly GAG→GGGArg255Ser CGC→AGC Ala290Val GCA→GTA Ala295Thr GCA→ACA Leu353Val CTA→GTAGln397Gln* CAG→CAA ix139-37 Val78Ala GTA→GCA Phe107Phe* TTC→TTTAsn159Asn* AAC→AAT Thr175Ile ACA→ATA Ala184Val GCA→GTA Asn186Asp AAC→GACArg203Arg* CGC→CGT Asp217Val GAT→GTT His236Gln CAT→CAG Glu252Gly GAG→GGGAla290Val GCA→GTA Leu353Val CTA→GTA Gly396Met GGT→AGT Thr427Thr* ACA→ACTix139_43 Glu4Glu* GAA→GAG Val78Ala GTA→GCA Phe107Phe* TTC→TTT Thr175IleACA→ATA Ala184Val GCA→GTA Ser226Ile AGC→ATC His236Gln CAT→CAG Glu252GlyGAG→GGG His266His* CAC→CAT Ala290Va1 GCA→GTA Leu353Val CTA→GTA *Silentmutation “N”: Nucleotide is unclear “?”: Amino acid is unclear

Evolved P450 BM-3 Variants Hydroxylate Various Substrates

The chosen screen was sensitive to hydroxylation of the methyleneadjacent to the oxygen atom of the surrogate the p-nitrophenoxyoctanesubstrate. Activity towards octane, hexane, and cyclohexane wastherefore measured, by measuring the rate of NADPH oxidation, which wasassumed to be fully coupled to alkane oxidation. As a result, relativeactivities determined using NADPH oxidation were assumed to equal therelative activities on the different substrates.

Compared to wildtype, for octane oxidation, the k_(cat) for IX139-3improved 50 times, while the K_(m) increased 45-fold (FIG. 5). Thewildtype kinetic values for hexane and cyclohexane were of the samemagnitude as for octane.

Results showing that hexane and cyclohexane were oxidized by IX79_(—)1were verified with GC/MS. Samples containing the cell lysate ofIX79_(—)1, hexane, and NADPH produced 2- and 3-hexanol (FIG. 6).1-Hexanol was not detected. No products were found with IX79_(—)1 andhexane alone, as a control.

IX79_(—)1 in the presence of cyclohexane and NADPH producedcyclohexanol, and cyclohexanone was not detected (FIG. 7). Again, noproducts were detected with IX79_(—)1 and cyclohexane alone.

Results also showed that IX139-3 was able to oxidize propane and butane,as verified with GC/MS. The reaction mixture contained enzyme, propaneor butane, and in the presence of NADPH produced 2-propanol and2-butanol (FIG. 8), respectively. Terminal hydroxylation was notdetected under these reaction conditions.

Since the activity towards medium chain alkanes had already reached asignificant level in generation five and IX139-3 displayed detectableactivity for propane oxidation, it was decided to include propane intothe screening program in the sixth generation. NADPH consumption assaysof the sixth generation showed that mutant IX139-3 and some new variantsalso oxidized propane.

Agar Plate Colony Assay for Screening Mutant Libraries

This assay was based on the formation of the purple dye formazan uponreaction of NADPH with Nitro Blue Tetrazolium (NBT) salt in presence ofthe catalyst, phenazine methosulfate (oxidized, PMS) This color reactionis known as a photometric standard assay for dehydrogenase activityresulting in reduction of their cofactor NADP+ to NADPH.

In the present investigations it was used to measure depletion of NADPHthat accompanies the P450-catalyzed oxidation of substrate. Bacterialcolonies which are locally using up the cofactor from filter papersoaked with NADPH and substrate for hydroxylation reactions remainedwhite after reaction of the remaining NADPH in the filter by reactionwith NBT. This screen resulted in white spots on a purple filter papercaused by bacterial colonies consuming NADPH. The screen will be usedfor prescreening libraries of future generations. Positive clones couldbe verified with the other assay systems that look directly at oxidizedproduct formation, since clones showing strongly uncoupled NADPHconsumption, and no substrate oxidation, might appear as improvedhydroxylation variants (false positives).

Example 2 Directed Evolution of a Cytochrome P450 Monooxygenase forOrganic Solvent Resistance

The total activity of P450 BM-3 in 96-well plates is relatively low,especially in the presence of an organic solvent that further reducesthe fraction of active enzymes. The mutant F87A converts the 12-pNCAsubstrate 4-5fold faster than the wild-type, the K_(m) value is 1.5-foldlower, and the chromophore from 2-pNCA substrate is released completelyand not only to 33% (Farinas, 2001; Schwaneberg, 1999a). Therefore, theF87A mutant and not the wildtype was used as a starting point of the invitro directed evolution. The evolutionary experiment to discover moreorganic solvent resistant variants was performed under very restrictiveconditions in order to preserve the valuable properties of the parents,a for monooxygenases remarkably high total activity and thermostability.The thermostability was under selective pressure by using thetemperature inducible PRPL-promoter system, and only clones that showeda high activity and a high organic solvent resistance were used asparents for further generations.

Experimental

All chemicals were of analytical reagent grade or higher quality andwere purchased from Fluka, Sigma or Aldrich. THF (Aldrich, 99.9%) andDSMO (Mallinckrodt AR, 99.9%) were of highest available purity grade.Enzymes were purchased from New England Biolabs, Stratagene, andBoehringer Mannheim.

Cultivation and Expression in 96-Well Plates

The P450 BM-3 and P450 BM-3 F87A genes are under the control of thestrong temperature inducible PRPL-promoter. Mutated BM-3 F87A variantswere cloned into the pUSCI BM-3 vector by using BamHI//EcoRI orAge//EcoRI restriction sites. Transformed clones grown on LBamp plates(Genetix) were transferred via a colony picker (QPix; Genetix) into 96well plates (flat bottom; Rainin) containing 120 μl LB culturesupplemented with 12 μg ampicillin per well. After growth for 12 hoursat 37° C. in a shaked incubator (280 rpm) 3 μl of each culture wastransferred with a grooved 96-pin deep-well replicator tool(V&P-Scientific) into 2 ml deep-well plates (Becton Dickinson)containing 400-500 μl of enriched TBamp medium. TBamp solution wassupplemented with 75 μl trace element solution (0.5 g CaCl₂×2H₂O, 0.18 gZnSO₄×7H₂O, 0.10 g MnSO₄, H₂O, 20.1 g Na_(e)-EDTA, 16.7 g FeCl₃×6H₂O,0.16 g CuSO₄×5H₂O, 0.18 g CoCl₂×6H₂O, add 1 L H₂O and autoclaVe) and 2mg aminolaevulinic acid per 50 ml TB. E. coli cells were grown in thismedium for 6 h at 37° C. then induced for 14 h at 42° C. All deep-wellplates were covered with a taped lid. The original LBamp plates werestored until further use at 80° C. after adding 100 μl glycerol(sterile, 50% (v/v)).

Screening Procedures

All experiments were performed in organic solvent resistantpolypropylene flat bottom 96 well plates (Greiner Bio-one). From eachplate a blank pre-reading was recorded prior performing assayprocedures.

Fast prescreen procedure. Deep well cultures were mixed well by a liquidhandling machine (Multirnek 96; Beckman) and 90 μl cell culture wastransferred to each well of the reference and assay plate. To each well40 μl Tris/HCl buffer (25 mM; containing 200 μM polymyxin B) and 5 μl12-pNCA (15 mM, dissolved in DMSO) were pipetted using the Multimek. Inaddition 4.5 μl THF or 45 μl DMSO were transferred to each well of theassay plate. After an incubation time of 12 min the 12-pNCA conversionis initiated by adding 20 μl of a Isocitric co-factor regenerationsolution (Isocitric acid 20 mM; dH₂O, NADP⁺ 3 mM, Isocitricdehydrogenase 0.8 U/ml). The reaction was stopped after visible colordevelopment by the addition of 50 μl NaOH (1.5 M). After 8-12 hincubation and removal of the bubbles using the Bunsen burner theabsorption at 410 nm of the clear solution was recorded. The P450 BM-3variants revealing a high activity and a high organic solvent resistancewere used for rescreening.

Rescreen procedure. Deep well cultures were in contrast to theprescreening method centrifuged at 4000 rpm for 10-20 min to remove thebrownish TB media. The cell pellets were frozen overnight at 20° C. andresuspended in 200 μl lysomix (pH 7.5, 25 mM Tris/HCl or 25 mM K_(x)PO₄supplemented with 1-50 mg lysozyme (Sigma) per 100 ml). 90 μl cellsuspension per well was transferred to the reference plate and the assayplate. To lyse the E. coli cells the plates were incubated at 37° C. for1 h. To each well 30 μl Tris/HCl buffer (25 mM) and 5 μl 12-pNCA (15 mM,dissolved in DMSO) were pipetted using the Multimek96. 15 μl THFsolution (15% (v/v); ddH₂O) were additionally added to each well of theassay plate. After an incubation time of 12 min the 12-pNCA conversionis initiated by adding 20 μl of a Isocitric co-factor regenerationsolution (Isocitric acid 20 mM; dH₂O, NADP+ 3 mM, Isocitricdehydrogenase 0.8 U/ml). The reaction was stopped after visible colordevelopment by the addition of 100 μl UT-buster (NaOH 1.5 M, 1.5 M Urea,50% (v/v) DMSO). After 8-12 h incubation and removal of the bubblesusing the Bunsen burner the absorption at 410 nm of the clear solutionwas recorded. The P450 BM-3 variants revealing a high activity and ahigh organic solvent resistance were cultured and expressed in shakingflasks for further characterization.

Shaking Flask Cultures and Purification

Fifty and 500 ml cultures were inoculated with a 1:100 dilution of anovernight Luria-Bertani (LB) culture of recombinant E. coli DH5containing the pT-USC1 BM-3 variant. The cells were shaken at 300 rpm at37° C. At an OD578=0.8-1 the cells were induced by increasing thetemperature to 42° C. After 8 h, the cells were harvested bycentrifugation at 4-8° C. The cell pellet was resuspended in Tris-HCl(15 ml, 0.1 M, pH 7.8) and lysed by sonication (3×2 minutes; outputcontrol=5, duty cycle 40%; Sonicator, Heat Systems—Ultrasonic, Inc.).The lysate was centrifuged at 23,300 g for 30 min. The supernatant wasfurther cleared through a low protein binding filter (0.45 μM). Thefiltrate was loaded on a SuperQ650M anion exchanger column (TosoHaas)and purified as previously described (Schwaneberg, 1999b).

Photometric Enzyme Assays

All photometric assays were carried out under aerobic conditions. UV/vismeasurements were performed in a Shimadzu spectrophotometer(BioSpec-1601). P450 BM-3 F87A concentrations were measured byCO-difference spectra, as reported by Omura and Sato using ε=91 mM⁻¹cm⁻¹ (Omura and Sato, 1964). Conversion of the p-nitrophenoxydodecanoicacid (12-pNCA) was monitored at 410 nm using a ThermomaxPlus platereader (Molecular Devices) and an ε=13,200 M⁻¹ cm⁻¹ (Schwaneberg et al.,1999a). The principle of the p-nitrophenoxycarboxylic acid (ANCA) assaysystem is described in FIG. 9. ω-Hydroxylation of pNCA by P450 BM-3leads to an unstable hemiacetal intermediate, which spontaneouslydissociates into the ω-oxycarboxylic acid and the yellow chromophorep-nitrophenolate. This assay system allows a continuous photometricdetection of the P450 BM-3 activity, as measured by the maximum turnoverrate, i.e., the number of product molecules generated per minute(Schwaneberg et al., 1999a).

TABLE 5 PCR Primers. “N”means that any nucleotide (A, T, G, or C) can be used. DesignationSequence (5′→3′) SEQ ID NO: For error-prone PCR pTBamHdGAA CCG GAT CCA TGA CAA TTA AAG AAA TGC 13 Rev3250CTA TTC TCA CTC CGC TGA AAC TGT TG 14For saturation mutagenesis at hot positions: pT235_FGCG ATG ATT TAT TAN NNC ATA TGC TAA ACG GA 15 pT235_RTCC GTT TAG CAT ATG NNN TAA TAA ATC ATC GC 16 pT471_FCAG TCT GCT AAA AAA GTA NNN AAA AAG GCA 17 GAA AAC GC pT471_RGCG TTT TCT GCC TTT TTN NNT ACT TTT TTA GCA 18 GAC TG pT1024_FGAC GTT CAC CAA GTG NNN GAA GCA GAC GCT 19 CGC pT3074_RGCG AGC GTC TGC TTC NNN CAC TTG GTG AAC GTC 20For back-mutation of F87 position: A87F1GCA GGA GAC GGG TTA TTT ACA AGC TGG ACG 21 A87F2CGT CCA GCT TGT AAA TAA CCC GTC TCC TGC 22 F87Gly1GCA GGA GAC GGG TTA GGT CAA GCT GGA CG 23 F87Gly2CGT CCA GCT TGT ACC TAA CCC GTC TCC TGC 24 F87Trp1GCA GGA GAC GGG TTA TGG ACA AGC TGG ACG 25 F87Trp2CGT CCA GCT TGT CCA TAA CCC GTC TCC TGC 26 F87His1GCA GGA GAC GGG TTA CAC ACA AGC TGG ACG 27 F87His2CGT CCA GCT TGT GTG TAA CCC GTC CTC CTG C 28 F87Asn1GCA GGA GAC GGG TTA AAC ACA AGC TGG ACG 29 F87Asn2CGT CCA GCT TGT GTT TAA CCC GTC TCC TGC 30 F87Asp1GCA GGA GAC GGG TTA GAT ACA AGC TGG ACG 31 F87Asp2CGT CCA GCT GTA TCT AAC CCG TCT CCT GC 32 F87Arg1GCA GGA GAC GGG TTA CGT ACA AGC TGG ACG 33 F87Arg2CGT CCA GCT TGT ACG TAA CCC GTC TCC TGC 34 F87Val1GCA GGA GAC GGG TTA GTT ACA AGC TGG ACG 35 F87Val2CGT CCA GCT TGT AAC TAA CCC GTC TCC TGC 36 F8711e1GCA GGA GAC GGG TTA ATT ACA AGC TGG ACG 37 F8711e2CGT CCA GCT TGT AAT TAA CCC GTC TCC TGC 38 F87Lys1GCA GGA GAC GGG TTA AAA ACA AGC TGG ACG 39 F87Lys2CGT CCA GCT TGT TTT TAA CCC GTC TCC TGC 40

Mutgenesis Conditions

For all the PCR reactions the thermocycler PTC 200 (MJ Research) wasemployed.

Protocol 1: First Mutant Generation

Component Volume (μl) ddH2O 42. 

 X -Y Buffer (10X) 5 dNTP (10 mM) 1 pT_USC1 BM-3 1 (of a mini-prep) RO X(27 pmol) Rev3250 Y (27 pmol) MnCl₂ 0.04 mM Taq polymerase 5 U Totalvolume 50

-   -   PCR program: 94° C. for 4 min        -   94° C. for 1:10 min; 55° C. for 1:30 min 72° C. for 4 min            (30 cycles)        -   72° C. for 10 min (1 cycle)

Protocol 2 (Gene Morph Kit): Second Mutant Generation

Components Volume (μl) ddH₂O 40. 

 X -Y Buffer 10X (Provided in kits) 5 dNTP mix (Provided in kits) 1Plasmid 2.5 pT_BamHI X (20 pmol) Rev3250 Y (20 pmol) Mutazyme (Providedin kits) 1 Total volume 50

-   -   PCR program: 95° C. for 30 s (1 cycle)        -   95° C. for 30 s; 55° C. for 30 s; 72° C. for 3:30 min (30            cycles)        -   72° C. for 10 min (1 cycle)

Protocol 3: Site Directed and Saturation Mutagenesis

Components Volume (μl) ddH2O 4 

 X Y Pfu Buffer (10x) (From Stratagene) 5 Plasmid (1:20 dilution) 2 dNTPmix (10 mM) 1 Forward primer X (17.5 pmol) Reverse primer Y (17.5 pmol)Pfu turbo (From Stratagene) 1 Total volume 50

-   -   PCR program: 94° C. for 4 min (1 cycle)        -   94° C. for 1:15 min; annealing 1:15 min; 68° C. for 16 min            (20 cycles)        -   68° C. for 20 min (1 cycle)    -   Annealing temperature: 55° C. for pT235, pT471 and pT102        -   60° C. for back-mutations at position 87

Mutagenesis and Results

The results of these experiments are shown in TABLES 6 and 7 and FIGS.10-13, and discussed below.

TABLE 6 Selected Cytochrome P450 Mutants Created From BM-3F87A by Directed Evolution and other techniques.All mutations are relative to the wild-type cyto-chrome P450 BM-3 (SEQ ID NO: 2, and only non-silent mutations are shown. Generation/ Amino Acid Codon Mutation StepMutant Mutation Mutation — F87A F87A TTT→GCA 1st Generation F87AB5 F87ATTT→GCA T235A ACG→GCG S1024R AGT→AGA F87APEC3 F87A TTT→GCA R471C CGC→TGCSaturation F87ASB3 F87A TTT→GCA mutagenesis R471A CGC→GCT T235A ACG→GCGS1024R AGT-AGA F87ABC1F10 F87A TTT→GCA R1024T AGA→ACG T235A ACG→GCGR471A CGC→GCT F87ABC1B6 F87A TTT→GCA R1024K AGA→AAA T235A ACG→GCG R471ACGC→GCT 2nd Generation F87A5F5 F87A TTT→GCA R471A CGC→GCT E494K GAA→AAAR1024E AGA→GAG T235A ACG→GCG Back-mutation Wd A87F GCA→TTT W5F5 R471ACGC→GCT E494K GAA→AAA R1024E AGA→GAG T235A ACG→GCG WSB3 R471A CGC→GCTT235A ACG→GCG S1024R AGT→AGA WB5 T235A ACG→GCG S1024R AGT→AGA WBC1F10R1024T AGA→ACG T235A ACG→GCG R471A CGC→GCT Site-directed F87G F87GGCC→GGT mutagenesis F87A F87A GCC (starting point) F87V F87V GCC→GTTF87I F87I GCC→ATT F87W F87W GCC→TGG F87D F87D GCC→GAT F87N F87N GCC→AACF87H F87H GCC→CAC F87K F87K GCC→AAA F87R F87R GCC→CGT

TABLE 7 Relative increase in 12-pNCA specific activity in the absence ofadditional co-solvents for 12-pNCA (“Absence of Co-Solvents”); andrelative increases in total activity at 10% (v/v) DMSO (“10% DMSO”) or2% (v/v) THF (“2% THF”). P450 BM-3 Variant Absence of Co-Solvents 10%DMSO 2% THF F87A 1.00 1.0 1.0 F87A5F5 3.40 5.5 10.0 F87ASB3 2.53 3.7 5.7F87AB5 2.85 3.7 5.3 F87ABC1F10 2.87 4.4 7.9 Wd 1.00 1.0 1.0 W5F5 2.515.9 3.4 WSB3 1.46 3.1 1.7 WB5 1.03 1.8 1.0 WBC1F10 2.01 3.5 2.3

First Mutant Generation

Random mutations were introduced by PCR into the BM-3 F87A gene codingfor 1049 amino acids and a His6 tag at the C-terminal end, underconditions designed to generate an average of one to two amino acidsubstitutions per gene (protocol 1). The mutant library was screened forclones with improved organic solvent resistance by comparing the,activity in the presence and absence of a co-solvent. Approximately6,520 clones were tested in 96 well plates using the 12-pNCA assay inpresence and absence of an organic solvent. The candidates with hightotal activity and high activity ratios (activity in presence divided byactivity in absence of an organic solvent) were selected forre-screening (protocol 1). Positive results of these assays wereverified after expressing 39 clones of the first generation in 500 mlscale. Many of these clones were expressed higher than F87A. Afterlysing the E. coli cells and determining the P450 content, the organicsolvent activities were measured using 1 ml cuvettes. The percentage ofthe relative activity in presence of organic solvent divided by theactivity in absence of a co-solvent of these 39 clones is shown in FIG.10. For more than 50% of the re-screened clones a superior organicsolvent resistance was found. Interestingly, apart from false positivestwo types of clones were discovered a) Increased DMSO and reduced THFresistance, b) Increased DMSO and increased THF resistance. No cloneswith increased DMSO and reduced THF resistance were detected. For ourpurposes, the mutants with multiple organic solvents resistances are themost interesting ones. The in DMSO most resistant mutants F87AB5 andF87APEC3 were selected for detailed analysis. Sequencing revealed threenon-silent mutations, two in F87AB5 (T(ACG)235A(GCG); S(AGT)1024R(AGA))and one in F87APEC3 (R(CGC)471C(TGC)). Interestingly, position 471 wasagain mutated in another clone, a substitution to serine instead ofcystein was found. Under the assumption of identical expression levelsan increased total activity of 3.7-fold at 10% (v/v) DMSO and of5.3-fold at 2% (v/v) THF was measured for F87AB5 (Table 7).

Saturation Mutagenesis (SM)

Only a limited set of amino acid (aa) substitutions can be explored byPCR mutagenesis at low error-rates and many as substitutions thatrequire the exchange of two or more nucleotides will not be present inthese PCR libraries. SM at sites identified by error-prone PCR allowsexploring these as changes and can result in the discovery of superiorcatalysts. Therefore, SM was used to introduce all 20 as into positionto the double mutant B3. Screening of about 576 clones revealed a moreactive triple mutant SB3. When expressed and purified in parallel withF87A, F87ASB3 revealed a up to 4 times higher expression level than F87Aand the DMSO and THF resistance was further improved as shown in FIGS.11A, 11B and by the organic solvent resistance profile in FIG. 12A.Sequencing revealed an exchange of R(CGC)→471A(GCT). The SM of theposition A235 of clone SB3 resulted in no further improvements. All 5sequenced clones contained an alanine at α-position 235. SM at position81024 revealed two more active clones, F87ABCIFIO and F87ABCIB6.F87ABCEF10 contains a R(AGA)1024T(ACG) substitution and BC1B6 aR(AGA)1024K(AAA) substitution. A detailed analysis of F87ABCIFIOrevealed increased total activity of 4.4 fold at 10% (v/v) DMSO and of7.9-fold at 2 (v/v) THF compared to the grandparent F87A (Table 7).

Second Mutant Generation (GeneMorph Kit)

In parallel to the saturation mutagenesis, random mutations wereintroduced into the F87ASB3 clone. The Taq-Polymerase used in the firstround of error-prone PCR has a strong bias to transitions whereas thepolymerase in the GeneMorph is biased toward transversions (StratageneInc.). This round of error-prone PCR should therefore result in adifferent set of mutation and as changes. After only screening about1440 clones, the mutant F87A5F5 was found. Sequencing revealed twonon-silent transversions at α-positions E(GAA)494K(AAA) andR(AGA)1024E(GAG). The simultaneous exchange of three nucleotides to thecomplementary one at position 1024 seems to be very unlikely, however ithas been confirmed by double sequencing this position in clone F87ASB3and F87A5F5. F87ASF5 revealed a higher organic solvent resistance forDMSO and THF than any previous clones. For F87A5F5 a, compared to F87A,increased total activity of 5.5-fold at 10% (v/v) DMSO and of 10-fold at2% (v/v) THF (Table 7) was discovered. The mutagenic pathways startingfrom the parent F87A are summarized in FIGS. 11A and 11B and the organicsolvent resistance profiles in DMSO, THY, acetone, acetonitrile, DMF,and ethanol are shown in FIGS. 12 and 13. In particular, FIGS. 12A-Gshow that the evolved mutants, including F87A5F5, exhibit an increasedresistance to these organic solvents.

Purification

F87A, F87ASB3 and the wild-type were simultaneously expressed and one byone purified (FIG. 12C). A comparison of the organic solvent resistancebetween lysed crude extracts and purified monoxygenase revealed verysimilar resistance toward DMSO (FIGS. 12C and 13A). For THF, theresistance of the purified enzyme was reduced between 3-9%. Thisreduction might be correlated to hydrophobic impurities in crude proteinextracts and relatively small amounts of THF present in the reactionsolution.

Back-Mutation of Position 87

The comparison of the organic solvent activity of the wild-type and themutant F87A revealed a significant higher resistance of the wild-typeenzyme in organic co-solvents (FIG. 12C). Therefore, the best clones ofthe laboratory evolution experiment (FIG. 3) were back-mutated atposition 87 to the wild-type. FIGS. 12B and 13B show that the activityprofile of the fraction of in organic solvent active clones isespecially for DMSO shifted to higher co-solvent concentrations. Forexample, the back-mutated clone W5F5 has an increased total activity of5.9-fold at 25% (v/v) DMSO and of 3.4 fold at 2 (v/v) THF compared tothe wild-type (Table 7). However, improvements are generally for thewild-type mutants, especially in the case of THF, lower compared to thefactors for F87A. Interestingly, 30% (v/v) DMSO seems to be a thresholdvalue to trigger a reduced organic solvent resistance. However, theexpression rates and activity of W5F5 are sufficient to use this mutantas a starting point for further directed evolution.

The results of the back-mutation encouraged us to investigate in detailthe influence of position 87 toward the activity of P450 BM-3 in organicco-solvents. By site-directed mutagenesis as changes from F to G, A, V,I, F, W, D, N, H, K and R were introduced (protocol 3) and confirmed bysequence analysis. The as changes to A, F revealed a fast 12-pNCAconversion and to I, V, G a lower one. All other mutants showed nodetectable activity for 12-pNCA. These results were confirmed by usingcoumarone as a substrate and the Gibbs assay to detect hydroxylatedproducts. The only exception was a low activity of the F87H towardcoumarone. Initial analysis of the active clones discovered asize-depending organic solvent resistance in the order F>I>A, G, V.

Evolution has generated a stunning variety of enzymes throughmutation/recombination and natural selection. However, monooxygenasesare not well suited for industrial application. The results reportedherein proves that laboratory evolution offers a fast and elegant way toadapt these enzymes to our needs in biotechnology applications. Theachieved improvements in organic co-solvents resistance will bring thisexceptional class of enzymes a step closer to industrial applications.

Example 3 Investigations of Solvent Conditions and Substrates for P450BM-3 Mutant IX139-3

Cytochrome P450 BM-3 from Bacillus megaterium (Bodupalli et al., 1990)(P450 BM-3), a medium-chain (C12-C18) fatty acid monooxygenase, has beenconverted into a highly efficient catalyst for the conversion of alkanesto alcohols (See Example 1). The evolved P450 BM-3 exhibits higherturnover rates than any reported biocatalyst for selective oxidation ofhydrocarbons. Unlike naturally-occurring alkane hydroxylases, among thebest known of which are the large, membrane-associated complexes ofmethane monooxygenase (MMO) and AlkB, the evolved enzyme is watersoluble and does not require additional proteins for catalysis. Theevolved alkane hydroxylase was found to have even higher activity onfatty acids, the presumed biological substrates for P450 BM-3, which wasalready one of the most efficient P450s known. A broad range ofsubstrates that includes the gaseous alkane propane induces the low tohigh spin shift, which activates the enzyme. The first soluble catalystfor alkane hydroxylation at room temperature, this laboratory-evolvedP450 opens new opportunities for clean, selective, hydrocarbonactivation for chemical synthesis and bioremediation.

Materials and Methods Expression of P450 BM-3 Variants

See Example 1.A. Expression and purification of P450 BM-3 variants.

Mutagenic PCR and StEP Recombination

For the first two generations, mutagenic PCR of the heme domain wasperformed as described (Farinas et al., 2001), using the followingprimers together with Taq polymerase (Roche):

(SEQ ID NO: 41) Bamfor 5′-ACAGGATCCATCGATGCTTAGGAGGTCATATG-3′(SEQ ID NO: 42) Sacrev 5′-GTGAAGGAATACCGCCAAG-3′

The PCR product was cloned by replacing the BamHI/SacI fragment of pBM-3_WT18-6. Nine mutants from generation 2 showing at least 2-foldimproved activity on 8-pnpane were recombined by staggered extensionprocess (StEP) (see Example 1; Zhao et al., 1998) using the same primersand 10 seconds extension time. A variant with 3 mutations (V78A, H236Q,E252G) with at least 2-fold improvement in activity relative to theparents was isolated. The 4th and 5th generations were generated byerror-prone PCR using the Genemorph kit (Stratagene) according to themanufacturer's protocol, using approximately 1 to 10 ng of template DNA.The most active mutant, IX139-3, was isolated from the 5th generation.Sequencing of the gene revealed 13 point mutations. Eleven lead to aminoacid substitutions (V78A, F107F, H138Y, T175I, V178I, A184V, H236Q,E252G, R255S, A290V, A295T, L353V, Q397Q), and two were synonymous.

Preparation of Cell Lysates

For high throughput screening, clones from the first three generationswere cultivated as described (See Example 1; and Farinas et al., 2001).For subsequent generations, colonies were picked and inoculated by aQpix (Genetix) robot into Luria Bertani media (LB, 350 μL, 100 mg/Lampicillin) into 1 mL deep well plates. The plates were incubated at 30°C., 250 rpm, and 80% relative humidity. After 24 hours, clones from thispre-culture were inoculated using a 96 replicator pin into 2 ml deepwell plates containing Terrific broth media (TB, 400 μL), ampicillin(100 mg/L), isopropy-β-D-thiogalactoside (IPTG, 10 μM), and ALA (0.5mM). The clones were cultivated at 30° C. for 24-30 hours. Cell pelletswere frozen at −20° C. and resuspended in phosphate buffer (1 mL, 0.1 M,pH 8.0) containing lysozyme (0.5 mg/mL), DNase I (0.1 μg/mL) and MgCl₂(10 mM). After 60 min at 37° C., the lysates were centrifuged and thesupernatant was diluted for activity measurements in 96 well microtiterplates.

High Throughput Determination of Enzymatic Activity

Mutant libraries were screened on 8-pnpane as described (See Example 1and Farinas et al., 2001). A cofactor (NADPH) depletion assay was usedto determine turnover rates. E. coli lysates of the mutants were dilutedinto 96 well microtiter plates containing phosphate buffer (200 μl, 0.1M, pH 8.0), alkane substrate (0.5-1 mM), and DMSO (1%). The liquidalkanes were added to the buffer using alkane stock solutions in DMSO,whereas the gaseous alkanes were bubbled into buffer for about 45 min toobtain saturated solutions. The reaction was initiated by addition ofNADPH (200 mM), and the oxidation of NADPH was monitored at 340 nm. Atotal of about 10,000 colonies were screened over 5 generations. Thedetermination of the maximal turnover rate is described in Example 1.

Solid Phase Assay

A solid phase NADPH depletion assay was used preselection of thefifth-generation mutant library. Cells were grown on LB agar platescontaining ampicillin (100 μg/ml), ALA (1 mM) andisopropyl-β-d-thiogalactopyranoside (10 μM).

The assay solution contained phosphate buffer (0.1 M, pH 8.0), polymyxinB sulfate (100 μM) as a cell permeabilizer, NADPH (2 mM), and substrate(5 mM) and was sonicated before use. A nitrocellulose membrane soakedwith this substrate solution was placed directly onto the colonies onthe agar plate. The sensitivity of the assay was regulated by the NADPHconcentration. After the reaction (about 5-15 min), nitro bluetetrazolium (0.5 mg/ml) in phosphate buffer (0.1 M, pH 8.0) and somecrystals of phenazine methosulfate were pipeted directly onto themembrane. Active colonies, which deplete NADPH, were identified as whitespots on the purple membrane. Positive colonies were picked with atoothpick and streaked out on fresh agar plates to obtain singlecolonies for rescreening.

GC/MS Analysis

See Example 1.

Substrate Binding

Dissociation constants for octane, hexane, and lauric acid weredetermined at 25° C. as described (Modi et al., 1995, herebyincorporated by reference in its entirety) from the change in absorptionat 418 nm upon substrate binding. For the alkanes, an enzyme solution(3-5 mM) in buffer (0.1 M potassium phosphate pH 8.0) was titrated witha stock solution of alkane (octane: 2 mM in methanol; hexane: 10 mM inmethanol). Methanol (1%) added to an enzyme solution does not induce aspin state shift. For laurate, the reaction solution contained enzyme(3-5 mM) and laurate (1 mM) in buffer (20 mM MOPS, 100 mM KCl, pH 7.4).Aliquots of the enzyme/substrate solution were removed and replaced withan equal volume of an enzyme solution.

Results

Table 8 shows the relative amounts of different products obtained foralkane oxidation, comparing wild-type cytochrome P450 BM-3 to theevolved mutant enzyme IX139-3.

TABLE 8 Product distribution for alkane oxidation by wild-type P450 BM-3and IX139-3 Substrate Product 139-3 (%) Wild-type (%) Octane 2-octanol66 17 3-octanol 32 40 4-octanol 2 43 Hexane 2-hexanol 19 20 3-hexanol 8180 Cyclohexane cyclohexanol 100 100 Butane 2-butanol 100 Not determinedPropane 2-propanol 100 Not determined

The results show that laboratory evolution methods consisting ofsequential rounds of random mutagenesis, in vitro recombination, andhigh throughput screening converted this highly efficient fatty acidmonooxygenase into one that can hydroxylate hexane and other alkanessimilarly well. In a preliminary study (Farinas et al., 2001), it wasverified that P450 BM-3 showed very low activity towards octane (Munroet al., 1993). A colorimetric screen using p-nitrophenoxy octane(8-pnpane) as an alkane substrate analog identified more active clones.Unfortunately, a plateau in the enzyme's activity on alkanes was reachedafter a few rounds of evolution, and further screening yielded no newimprovements.

When designing a screening strategy for identifying cytochrome P450 BM-3mutants that catalyze the hydroxylation of alkanes, it was necessary tohave an assay sensitive enough to observe improvements when theactivities were still very low. The colorimetric assay on the surrogatesubstrate 8-pnpane nicely fulfilled this role. The risk to using asurrogate substrate such as 8-pnpane, however, is that the enzyme maybecome ‘addicted’ to that particular substrate. Activity towards thedesired substrate may not be increasing in the same proportion (or notat all). By the third generation of mutagenesis and screening, the mostactive BM-3 variant acquired sufficient activity to enable us to screenmutant libraries for activity directly on an alkane (octane) using ahigh throughput NADPH consumption assay (see Example 1). NADPH oxidationalone, however, may not provide an accurate measure of catalyticactivity since reducing equivalents from NADPH can be diverted intoforming reduced oxygen intermediates (H₂O or H₂O₂). Therefore allsubsequent generations were screened using a combination of the 8-pnpaneassay, sensitive to product formation, and NADPH consumption in thepresence of octane (see Experimental).

By monitoring cell growth under conditions where the P450 enzyme wasexpressed, we also determined that the enzyme had become toxic to the E.coli cells during the process of acquiring higher activity towardsalkanes. This increased toxicity placed an artificial barrier on howactive the enzyme could become and still appear as a positive duringhigh throughput screening for alkane hydroxylase activity. By alteringthe growth and expression conditions to limit enzyme production duringgrowth we were able to continue the evolution and identify P450 BM-3mutants with very high alkane hydroxylation activities.

Five generations of mutagenesis and screening yielded P450 BM-3 mutantIX139-3. As shown in FIG. 14, the enzyme evolved using 8-pnpane andoctane to screen for more active clones was highly active on octane,hexane, cyclohexane and pentane. For example, IX139-3 is 38-fold moreactive on octane than the wildtype enzyme. The rates for hydroxylationof all the liquid alkanes exceed that of wildtype P450 BM-3 on its fattyacid substrates, lauric and palmitic acid (FIG. 14). The evolved enzymewas also 2-fold more active on palmitic acid (FIG. 14), which wassurprising since this fatty acid is presumably one of the natural, andoptimal, substrates for P450 BM-3. Analysis of the products of reactionwith n-alkanes showed hydroxylation at subterminal positions (Table 8),similar to wildtype enzyme's regioselectivity on fatty acids. No furtheroxidation to diols or ketones was observed. Cyclohexanol was the soleproduct of hydroxylation of cyclohexane. For the oxidation of octane,hexane, and cyclohexane, the ratio of products formed to dioxygenconsumed was 1:1, as determined by GC/MS, and H₂O₂ was not detected.This demonstrated that reducing equivalents derived from NADPH result insubstrate hydroxylation and the mutant does not waste electrons toproduce reduced oxygen intermediates. H₂O₂ is not detected for theoxidation of the remaining substrates, and it is assumed that substratehydroxylation is fully coupled to NADPH oxidation.

P450 BM-3 1×139-3 is a better catalyst than known, naturally-occurringalkane monooxygenases acting on their most preferred substrates (FIG.15). For example, the preferred substrate for the non-heme iron alkanemonooxygenase (AlkB) from Pseudomonas oleovorans is octane; its reportedmaximal turnover rate is 210 min-1 (Shanklin et al., 1997). In contrastto P450 BM-3, AlkB is membrane-bound and requires two additionalproteins, NADPH-rubredoxin reductase and rubredoxin, for catalyticactivity. Soluble (sMMO) and particulate methane monooxygenase (pMMO)are large (about 300 kDa), multimeric iron (and in the case of pMMO,iron-copper) enzymes with turnover rates of 200-250 min⁻¹ for gaseousalkanes (methane, 222 min⁻¹) (Fox et al., 1990; Fox et al., 1989; Tongeet al., 1977). Rates on alkanes larger than C4 are much lower (Green andDalton, 1989). Furthermore, AlkB and MMO have not been produced withhigh activity in heterologous hosts suitable for protein engineering(Shanklin 1997; Murrell et al., 2000; Staijen et al., 2000). Among thebest known of the cytochrome P450 alkane hydroxylases is P450 Cm1 (CYP52A3), which is involved in alkane metabolization in Candida maltosa(Zimmer et al., 1996). This enzyme is also membrane-bound, requires aseparate reductase, and has turnover rates lower than AlkB (27 min^(d)for the purified protein and 40 min⁻¹ for microsomal preparations)(Scheller et al., 1996). A number of other P450s also show low levels ofactivity for alkanes (Stevenson et al., 1996; Fisher et al., 1998; Munroet al., 1993).

From the intriguing possibility that the P450 BM-3 could be engineeredto accept the small, gaseous hydrocarbon substrates preferred by MMO,the ability of the IX139-3 mutant to hydroxylate butane and propane wasdetermined. Since the screen identified mutants more active on 8-pnpaneand longer-chain alkanes, high activities on propane and butane were notnecessarily expected. Based upon NADPH consumption, IX139-3 oxidizedbutane and propane with initial rates of 1800 and 860 min⁻¹,respectively (FIG. 14), which compared favorably to those of the muchlarger MMO. Comparing with the wildtype, IX139-3 hydroxylates butane andpropane 108 and 57 times faster, respectively. The sole products ofpropane and butane oxidation by P450 BM-3 1×139-3 were 2-propanol and2-butanol (Table 8).

The P450 resting state contains an iron protoporphyin IX as a low-spinsix-coordinate ferric species with a dissociable water ligated trans tothe proximal cysteinate (Ortiz de Montellano, 1995). Substrate bindingdisplaces water and generates a high-spin five-coordinate species. Thespin state shift causes the redox potential of the heme to increase,which activates the protein for hydroxylation. The heme's absorptionmaximum at 419 nm corresponds to the low-spin species; a shift to 390 nmis characteristic of the high-spin form. This spectral shift is inducedin IX139-3 by all the substrates (FIG. 16A), which allowed estimationsof K_(d)'s for octane, hexane, and laurate of 10 μM, 27 μM, and 19 μM,respectively. Only the fatty acid substrates produce a spin shift in thewildtype enzyme (laurate: K_(d)=260 μM).

Crystal structures of wildtype P450 BM-3 with and without substratereveal large conformational changes upon substrate binding at the activesite (Haines, D. C., Tomchick, D. R., Machius, M. & Peterson, J. A.Pivotal role of water in the mechanism of P450 BM-3. Biochemistry 40,13456-13465 (2001); Li, H. Y. & Poulos, T. L. The structure of thecytochrome p450BM-3 haem domain complexed with the fatty acid substrate,palmitoleic acid. Nat. Struct. Biol. 4, 140-146 (1997); Paulsen, M. D. &Ornstein, R. L. Dramatic Differences in the Motions of the Mouth of Openand Closed Cytochrome P450bm-3 by Molecular-Dynamics Simulations.Proteins 21, 237-243 (1995); Chang, Y. T. & Loew, G. Homology modeling,molecular dynamics simulations, and analysis of CYP119, a P450 enzymefrom extreme acidothermophilic archaeon Sulfolobus solfataricus.Biochemistry 39, 2484-2498 (2000)). The substrate free structuredisplays an open access channel with 17 to 21 ordered water molecules.Substrate recognition serves as a conformational trigger to close thechannel, which dehydrates the active site, increases the redoxpotential, and allows dioxygen to bind to the heme. Five of the 11 aminoacid substitutions in IX139-3 occur in the region which undergoes thelargest conformational change, the F and G helices and the loopconnecting them, as well as the I helix across which the F and G helicesmust slide (FIG. 4). The F and G helices serve as a lid which closesover the substrate access channel upon substrate binding.

Attempts to engineer catalyst specificity are often limited to alteringamino acids directly involved in substrate recognition and binding. Onlyone residue that is in direct contact with substrate in the wildtypeenzyme has been mutated in IX139-3 (V78A). Amino acids R47, Y51, F42,and F87 have been proposed to be essential for fatty acid substraterecognition (Noble et al., 1999). R47, Y51, and F42 are located at themouth of the substrate-binding pocket. R47 and Y51 interact with thesubstrate carboxylate moiety through electrostatic and hydrogen bondinginteractions, while F42 serves as a hatch covering the binding pocket.None of these important residues has been mutated in IX139-3. A saltbridge between R255 and D217 in the substrate free structure can bedisrupted by the R255S mutation in IX139-3. This mutation may facilitateconformational changes that permit alkanes to bind more favorably.Rational engineering of the substrate binding pocket of P450 BM-3produced a triple mutant (F87V, L188Q, A74G) with increased activity foroctane (Appel et al., 2001). Directed evolution to produce mutantIX139-3 did not find any beneficial mutations at these sites.

The fact that a few amino acid substitutions can produce a significantincrease in P450 BM-3's activity on fatty acids, the presumed biologicalsubstrates, indicates that natural selection does not place an advantageon maximizing activity, possibly because such a broadly active enzyme isalso toxic to the host organism, as it is to E. coli. By evolving theenzyme uncoupled from its biological context we are able to uncover thetrue catalytic potential of the cytochrome P450. P450 BM-3 mutantIX139-3 is the fastest alkane hydroxylase known. Easily over-expressedin E. coli, soluble and requiring no additional electron transferproteins for catalysis, this enzyme should prove an attractive catalystfor selective hydrocarbon oxidation.

Example 4 Alkene Oxidation for P450 BM-3 Mutant IX139-3

Epoxidation of alkenes is an important reaction in organic synthesissince they are important chemical building blocks. The oxirane ring issubject to ring opening by various nucleophiles (oxygen, sulfur,nitrogen, carbon), which yield bifunctional compounds (Carey andSundberg, 1990). In the chemical industry, epoxides are used in theproduction of polymers (polyether polyols), as well as glycols,polyglycols, and alkanolamines. Optically pure epoxides are usefulintermediates in the synthesis of pharmaceuticals, agrochemicals,perfumes, and liquid crystals where chirality plays a critical role infunction. Great efforts in developing chemical catalyst for alkeneepoxidation has resulted in limited successes (Jacobson, 1993; White etal., 2001). The main limitations of chemical methods for alkeneepoxidation is poor catalytic efficiencies for trans and terminalolefins (Faber, 2000). Furthermore, chemical methods produce largeamounts of toxic byproducts. For example, the current industrialprocesses for the synthesis of propylene oxide from propene use largeamounts of Cl₂ that lead to equipment corrosion and toxic byproducts.Monooxygenases provide an alternative to chemical means for epoxidation(Schmid et al., 2001).

The mutant IX139-3 was shown to have broad substrate specificity foralkanes with varying chain length (C8-C3). Furthermore, the variant wasalso shown to be more active on fatty acids. The activity for alkeneswas also investigated, and the mutant is also more efficient in alkeneoxidation. IX139-3 may prove to be a general catalyst for hydrocarbonoxidation, and it may find uses in the fine chemical industry as well asin bioremediation:

Materials and Methods

All chemical reagents were procured for Aldrich, Sigma, or Fluka.

Expression of P450 BM-3 Mutant

See example 1. A. Expression and purification of p450 BM-3 variants

Determination of the Maximum Initial Rate for Alkene Oxidation

The enzyme was purified and quantified as described above. A typicalreaction solution contained enzyme (1.0 ml, 1 μM), alkene (10 μL, 1.0mM), and methanol (1%) in potassium phosphate buffer (0.1 M, pH 8.0).The solution was incubated at room temperature, and the reaction wasinitiated by the addition of NADPH (200 μL, 200 μM). The rate of NADPHoxidation was monitored at 340 nm.

Substrate Conversion and Product Characterization

A typical reaction contained purified enzyme (1.0 ml, 1 μM), alkene (10μL, 1.0 mM), and methanol (1%) in phosphate buffer (0.1 M, pH 8.0). Thesolution was incubated at room temperature for 5 minutes, and thereaction was initiated by the addition of NADPH (200 μL, 200 μM). Thesolution was allowed to stir aerobically at room temperature for 30minutes. For propene oxidation, the reaction contained enzyme (3.0 ml,1.0 μM) in potassium phosphate buffer (0.1 M, pH 8.0), and the resultingsolution was sealed in a 20 ml vial with a septum. The head-space wasfilled with propene and the reaction was initiated by the addition ofNADPH (100 μl, 0.5 mM). The reaction was stirred at room temperature for1.5 hours.

The products were analyzed by gas chromatography/mass spectrometry usingan Hewlett Packard HP 6890 series gas chromatograph coupled with anHewlett Packard 5973 mass selective detector). The GC was fitted with aHP FFAP column (crosslinked FFAP, 30 m×0.25 mm×0.25 μm film thickness).The condition for propene is as follows: (1) 35° C. for 1.7 minutes. (2)35 to 200° C. at 20° C./min. (3) 200° C. for 1 minute. The condition forstyrene, cyclohexene, and 1-hexene is as follows: (1) 100° C. for 4.5minutes. (2) 100 to 200° C. at 20° C./min. (3) 200 for 7.0 minutes.Authentic standards were used to identify the products by retentiontime. Products were further verified by matching the fragmentationdistributions with a database in the software provided by the instrumentmanufacturer.

4-(1-Nitrobenzyl)pyridine Assay

A typical reaction contained enzyme (1.0 ml, 1×10⁻⁶ M) in potassiumphosphate buffer (0.1 M, pH 8.0), alkene (1.0×10⁻³ M), and methanol (1%)in a vial. The reaction was initiated with NADPH (50 μl, 1.0×10⁻³ M),and allowed to stir aerobically for 5 minutes. 300 μl of a stocksolution of 4-(4-nitrobenzyl)pyridine (5% w/w in acetone) was added tothe reaction, and the vial was sealed. The reaction was incubated at 80°C. for 20 minutes and chilled on ice. 600 μl of an ethylacetate/acetone(5:2) solution and 300 μl 5 M aqueous NaOH was added to the reactionsolution. The solution was mixed thoroughly, and the absorbance of theorganic layer was measured at 540 nm in a glass 96-well plate withmicroplate spectrophotometer.

Substrate Binding

See Example 3: Substrate binding.

Results and Discussion

P450 BM-3 is known to form epoxides from various substrates that vary insize and structure (Martinez and Stewart, 2000; Fruetel et al., 1994;Capdevila et al., 1996; Ruettinger and Fulco, 1981; Schneider et al.,1999). For example, P450 BM-3 oxidizes arachidonic acid to18(R)-hydroxyeicosatetraenoic acid and 14(S),15(R)-epoxyeicosatrienoicacid (80 and 20% of total products, respectively), and eicosapentaenoicacid to 17(S),18(R)-epoxyeicosatetraenoic acid (99% total products)(Capdevila et al., 1996). Furthermore, stryene is oxidized solely tostyrene oxide (Fruetel et al., 1994).

The evolved enzyme IX139-3 has been shown to be highly active for alkaneand fatty acid oxidation (see above). Interestingly, the initial ratesof NADPH consumption in the presence of alkenes were also very high(FIG. 17). For example, the rate of cyclohexene and styrene is 1200 and300 fold more active than wildtype, respectively. For all the alkenesinvestigated, H₂O₂ is not detected and the enzyme is assumed to be fullycoupled. However, there is a possibility that H₂O can be produced, whichcan only be accurately determined by quantitating product formation.

The products for styrene, propene, cyclohexene, and 1-hexene werecharacterized by gas chromatography/mass spectrometry. The sole productfor stryene was styrene oxide, which is similar to what is found for thewildtype (Martinez et al., 2000). The major product from propeneoxidation is propene oxide and allyl alcohol is only a minor species.Propene oxidation has not been previously reported by P450 BM-3 (SeeFIG. 18).

The products for styrene, propene, cyclohexene, and 1-hexene werecharacterized by gas chromatography/mass spectrometry. The sole productfor stryene was styrene oxide, which is similar to what is found for thewildtype (Fruetel et al., 1994). The products from propene oxidation ispropene oxide and allyl alcohol. Propene oxidation has not beenpreviously reported by P450 BM-3.

GC/MS analysis of cyclohexene oxidation by IX139-3 revealed twoproducts, cyclohexene oxide and 1,2-cyclohexane diol. The sole productof the bioconversion was most likely cyclohexene oxide, and1,2-cyclohexane diol probably occurred via base-catalyzed hydrolysis ofcyclohexene oxide (see reaction conditions) (March, 1992). Cyclohexeneoxide was converted to 1,2-cyclohexane diol in potassium phosphatebuffer (0.1 M, pH 8.0), as verified using GC/MS.

No epoxidation of 1-hexene was detected, and the product was1-hexene-3-ol, which results from hydroxylation at the allylic position.This was expected since wildtype P450 BM-3 preferentially oxidizes fattyacids with a terminal double bond at the ω-2 position and thecorresponding terminal epoxide is not formed (Shirane et al., 1993). Theselectivity may be due to the C—H bond strength. The ω-2 secondaryallylic (DH° 298˜83 kcal/mol) C—H are weaker than the ω-3 secondary (DH°298˜98 kcal/mol) C—H bond. However, a terminal double bond should alsobe oxidized unless there is a mechanistic or steric factor. P450 BM-3 isknown to form the corresponding epoxide from cis-9-hexadecenoic acid aswell as hydroxylated products (Ruettinger and Fulco, 1981), whichdemonstrates that there is no mechanistic restraint. Hence, a stericrestraint exist that hinders terminal epoxidation. This is furthersupported by the fact terminal oxidation of fatty acids and alkanes arenot observed.

The 4-(nitrobenzyl)pyridine (4-NBP) (Kim and Thomas, 1992) assay wasused to determine epoxide formation for allyl chloride, isoprene,2-hexene and 3-hexene since the corresponding epoxides were notavailable as standards. Nucleophilic attack of the oxirane ring by 4-NBPresults alkylation, and the product results in a purple colored(Abs_(max)˜550 nm) species. IX139-3 in the presence of isoprene,dioxygen and NADPH forms the alkylated product (FIG. 19) with aλ_(max)˜550 nm. Similar results occur when allyl chloride is thesubstrate. The color develops only when enzyme, substrate, and NADPH arepresent. If any of the components are missing then no color is formed.Control experiments with IX139-3, styrene, and NADPH, which is shown toform styrene oxide as determined by GC/MS, also test positive using the4-NBP assay. When either 2-hexene or 3-hexene are used as substrates, noalkylation product is formed, and it is assumed that the alkene ishydroxylated.

139-3 in the presence of alkenes induces small spin state shifts.Whereas, the wildtype in presence does not produce a spin state shift.This indicates that the substrate binding properties have been altered.IX139-3 is very active for alkane hydroxylation, fatty acid oxidationand alkene epoxidation, and the substrate specificity is broad. Thismutant may be useful a general catalyst for hydrocarbon oxidation. Themutant may be useful for fine chemical synthesis when only one productis detected such as styrene oxide. Alternatively, the mutant may findapplications in bioremediation when more than one product is generated.

GENERAL DEFINITIONS

The following defined terms are used throughout the presentspecification, and should be helpful in understanding the scope andpractice of the present invention.

“Cytochrome P450 monooxygenase” or “P450 enzyme” means an enzyme in thesuperfamily of P450 haem-thiolate proteins, which are widely distributedin bacteria, fungi, plants and animals. The enzymes are involved inmetabolism of a plethora of both exogenous and endogenous compounds.Usually, they act as oxidases in multicomponent electron transferchains, called here P450-containing monooxygenase systems. The uniquefeature which defines whether an enzyme is a cytochrome P450 enzyme isthe reduced form of the enzyme which binds carbon monoxide and resultsin a characteristic absorption maximum at 450 nm. Reactions catalyzed bycytochrome P450 enzymes include epoxidation, N-dealkylation,O-dealkylation, S-oxidation and hydroxylation. The most common reactioncatalyzed by P450 enzymes is the monooxygenase reaction, i.e., insertionof one atom of oxygen into a substrate while the other oxygen atom isreduced to water. Although any P450 enzyme can be modified according tothe invention, the following are non-limiting examples of preferred P450enzymes: P450 BM-3 (GenBank Accession Nos. J04832 (SEQ ID NO:1) andP14779 (SEQ ID NO:2)); CYP 2C3 (GenBank P00182, SEQ ID NO:3); CYP 2C9(GenBank P11712; SEQ ID NO:4), CYP 2D1v (GenBank P10633; SEQ ID NO:5),and CYP 108 (GenBank P33006; SEQ ID NO:6).

An “oxidation”, “oxidation reaction”, or “oxygenation reaction”, as usedherein, is a chemical or biochemical reaction involving the addition ofoxygen to a substrate, to form an oxygenated or oxidized substrate orproduct. An oxidation reaction is typically accompanied by a reductionreaction (hence the term “redox” reaction, for oxidation and reduction).A compound is “oxidized” when it receives oxygen or loses electrons. Acompound is “reduced” when it loses oxygen or gains electrons. Anoxidation reaction can also be called an “electron transfer reaction”and encompass the loss or gain of electrons (e.g., oxygen) or protons(e.g., hydrogen) from a substance. Non-limiting examples of oxidationreactions include hydroxylation (e.g., RH+O₂+2H⁺+2e⁻→ROH+H2O),epoxidation (RCH═CHR′+2H⁺+O₂+2e⁻→RCHOCHR′+H₂O), and ketone formation(RCH₂R′→RCOR′).

A “co-solvent” or “indirect solvent” herein means a non-solvent thatbecomes an acceptable solvent or co-solvent when a small amount ofactive solvent is added. For example, water is a non-solvent for varioushydrophobic substances, but the addition of a water-miscible solventsuch as DMSO, tetrahydrofuran (THF), methanol, ethanol, propanol,dioxane, or dimethylformamide (DMF), or other solvents known in the art,increases the solubility of hydrophobic compounds in the mixture.

The “organic solvent resistance” of an enzyme means its ability tofunction, optionally function over time, in an organic solvent or in aco-solvent. One way to evaluate organic solvent resistance is to assessthe ability of the enzyme to resist a loss of activity over time, in oneor more co-solvent systems. A more “organic-solvent resistant” enzymecan be one that is more resistant to loss of structure (unfolding) orfunction (enzyme activity) when exposed to an organic solvent orco-solvent. Preferred systems for testing organic solvent resistanceinclude water/DMSO and water/THF mixtures, for example, 10% (v/v) DMSOand 2% (v/v/) THF.

“Alkane-oxidation capability” herein means the capability of acytochrome P450 enzyme to oxidize at least one alkane. Thealkane-oxidation capability of an enzyme can be evaluated, for example,by mixing the enzyme with an alkane in the presence of any necessaryco-factors, and evaluate whether the alkane is oxidized. In particular,the capability of a cytochrome P450 variant to oxidize an alkane can berelated to the capability of the corresponding wild-type P450 to oxidizethe same alkane, e.g., by comparing maximum turnover rate, total amountof product formed, or any other suitable measure of enzyme activity.Many examples of alternative assays are provided herein. Preferredalkanes for which alkane-oxidation capability can be evaluated include8-pnpane, octane, hexane, cyclohexane, propane, ethane, and/or butane.

“Alkene-oxidation capability” herein means the capability of acytochrome P450 enzyme to oxidize at least one alkene. Thealkene-oxidation capability of an enzyme can be evaluated, for example,by mining the enzyme with an alkene in the presence of any necessaryco-factors, and evaluate whether the alkene is oxidized to form anepoxide or hydroxylated product. In particular, the capability of acytochrome P450 variant to oxidize an alkene can be related to thecapability of the corresponding wild-type P450 to oxidize the samealkene, e.g., by comparing maximum turnover rate, total amount ofproduct formed, or any other suitable measure of enzyme activity. Manyexamples of alternative assays are provided herein. Preferred alkenesfor which alkane-oxidation capability can be evaluated include octene,hexene, propene, ethene, and/or butene.

The term “about” or “approximately” means within an acceptable errorrange for the particular value as determined by one of ordinary skill inthe art, which will depend in part on how the value is measured ordetermined, i.e., the limitations of the measurement system. Forexample, “about” can mean a range of up to 20%, preferably up to 10%,more preferably up to 5%, and more preferably still up to 1% of a givenvalue. Alternatively, particularly with respect to biological systems orprocesses, the term can mean within an order of magnitude, preferablywithin 5-fold, and more preferably within 2-fold, of a value.

Molecular Biology Definitions

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature: See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (herein“Sambrook et al., 1989”); DNA Cloning: A Practical Approach, Volumes Iand II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gaited. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds.(1985)); Transcription And Translation (B. D. Haines & S. J. Higgins,eds. (1984)); Animal Cell Culture (R. I. Freshney, ed. (1986));Immobilized Cells And Enzymes (IRL Press, (1986)); B. Perbal, APractical Guide To Molecular Cloning (1984); P. M. Ausubel et al.(eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc.(1994).

A “protein” or “polypeptide”, which terms are used interchangeablyherein, comprises one or more chains of chemical building blocks calledamino acids that are linked together by chemical bonds called peptidebonds.

An “enzyme” means any substance, preferably composed wholly or largelyof protein, that catalyzes or promotes, more or less specifically, oneor more chemical or biochemical reactions. The term “enzyme” can alsorefer to a catalytic polynucleotide (e.g. RNA or DNA).

A “native” or “wild-type” protein, enzyme, polynucleotide, gene, orcell, means a protein, enzyme, polynucleotide, gene, or cell that occursin nature.

A “parent” protein, enzyme, polynucleotide, gene, or cell, is anyprotein, enzyme, polynucleotide, gene, or cell, from which any otherprotein, enzyme, polynucleotide, gene, or Cell, is derived or made,using any methods, tools or techniques, and whether or not the parent isitself native or mutant. A parent polynucleotide or gene encodes for aparent protein or enzyme.

A “mutant”, “variant” or “modified” protein, enzyme, polynucleotide,gene, or cell, means a protein, enzyme, polynucleotide, gene, or cell,that has been altered or derived, or is in some way different orchanged, from a parent protein, enzyme, polynucleotide, gene, or cell. Amutant or modified protein or enzyme is usually, although notnecessarily, expressed from a mutant polynucleotide or gene.

A “mutation” means any process or mechanism resulting in a mutantprotein, enzyme, polynucleotide, gene, or cell. This includes anymutation in which a protein, enzyme, polynucleotide, or gene sequence isaltered, and any detectable change in a cell arising from such amutation. Typically, a mutation occurs in a polynucleotide or genesequence, by point mutations, deletions, or insertions of single ormultiple nucleotide residues. A mutation includes polynucleotidealterations arising within a protein-encoding region of a gene as wellas alterations in regions outside of a protein-encoding sequence, suchas, but not limited to, regulatory or promoter sequences. A mutation ina gene can be “silent”, i.e., not reflected in an amino acid alterationupon expression, leading to a “sequence-conservative” variant of thegene. This generally arises when one amino acid corresponds to more thanone codon. Table 9 outlines which amino acids correspond to whichcodon(s).

TABLE 9 Amino Acids, Corresponding Codons, and Functionality Amino AcidSLC DNA codons Side Chain Property Isoleucine I ATT,ATC,ATA HydrophobicLeucine L CTT, CTC, CTA, CTG, TTA, TTG Hydrophobic Valine VGTT, GTC, GTA, GTG Hydrophobic Phenylalanine F TTT, TTCAromatic side chain Methionine M ATG Sulphur group Cysteine C TGT, TGCSulphur group Alanine A GCT, GCC, GCA, GCG Hydrophobic Glycine GGGT, GGC, GGA, GGG Hydrophobic Proline P CCT, CCC, CCA, CCGSecondary amine Threonine T ACT, ACC, ACA, ACG Aliphatic hydroxyl SerineS TCT, TCC, TCA, TCG, AGT, AGC Aliphatic hydroxyl Tyrosine T TAT, TACAromatic side chain Tryptophan W TGG Aromatic side chain Glutamine QCAA, CAG Amide group Asparagine N AAT, AAC Amide group Histidine HCAT, CAC Basic side chain Glutamic acid E GAA, GAG Acidic side chainAspartic acid D GAT, GAC Acidic side chain Lysine K AAA, AAGBasic side chain Arginine R CGT, CGC, CGA, CGG, AGA, AGGBasic side chain Stop codons Stop TAA, TAG, TGA —

“Function-conservative variants” are proteins or enzymes in which agiven amino acid residue has been changed without altering overallconformation and function of the protein or enzyme, including, but notlimited to, replacement of an amino acid with one having similarproperties, including polar or non-polar character, size, shape andcharge (see Table 9).

Amino acids other than those indicated as conserved may differ in aprotein or enzyme so that the percent protein or amino acid sequencesimilarity between any two proteins of similar function may vary and canbe, for example, at least 70%, preferably at least 80%, more preferablyat least 90%, and most preferably at least 95%, as determined accordingto an alignment scheme. As referred to herein, “sequence similarity”means the extent to which nucleotide or protein sequences are related.The extent of similarity between two sequences can be based on percentsequence identity and/or conservation. “Sequence identity” herein meansthe extent to which two nucleotide or amino acid sequences areinvariant. “Sequence alignment” means the process of lining up two ormore sequences to achieve maximal levels of identity (and, in the caseof amino acid sequences, conservation) for the purpose of assessing thedegree of similarity. Numerous methods for aligning sequences andassessing similarity/identity are known in the art such as, for example,the Cluster Method, wherein similarity is based on the MEGALIGNalgorithm, as well as BLASTN, BLASTP, and FASTA. When using all of theseprograms, the preferred settings are those that results in the highestsequence similarity.

The “activity” of an enzyme is a measure of its ability to catalyze areaction, i.e., to “function”, and may be expressed as the rate at whichthe product of the reaction is produced. For example, enzyme activitycan be represented as the amount of product produced per unit of time orper unit of enzyme (e.g., concentration or weight), or in terms ofaffinity or dissociation constants. Preferred activity units forexpressing activity include the catalytic constant (k_(cat)=v_(max)/E;Vmax is maximal turnover rate; E is concentration of enzyme); theMichaelis-Menten constant (K_(m)); and k_(cat)/K_(m). Such units can bedetermined using well-established methods in the art of enzymes.

The “stability” or “resistance” of an enzyme means its ability tofunction, over time, in a particular environment or under particularconditions. One way to evaluate stability or resistance is to assess itsability to resist a loss of activity over time, under given conditions.Enzyme stability can also be evaluated in other ways, for example, bydetermining the relative degree to which the enzyme is in a folded orunfolded state. Thus, one enzyme has improved stability or resistanceover another enzyme when it is more resistant than the other enzyme to aloss of activity under the same conditions, is more resistant tounfolding, or is more durable by any suitable measure. For example, amore “organic-solvent” resistant enzyme is one that is more resistant toloss of structure (unfolding) or function (enzyme activity) when exposedto an organic solvent or co-solvent.

The term “substrate” means any substance or compound that is convertedor meant to be converted into another compound by the action of anenzyme catalyst. The term includes aromatic and aliphatic compounds, andincludes not only a single compound, but also combinations of compounds,such as solutions, mixtures and other materials which contain at leastone substrate. Preferred substrates for hydroxylation using thecytochrome P450 enzymes of the invention include alkanes such aspropane, ethane, butane, pentane, hexane, cyclohexane, and octane, andalkane derivatives such as alkanes substituted with one or more chemicalgroup such as nitro-, sulphur-, halogen- and oxygen-containing groups,as well as aromatic groups, e.g., p-nitrophenoxyoctane (8-pnpane).Preferred substrates for epoxidation include alkenes such as propene,ethene, butene, pentene, hexene, cyclohexene, octene, as well as alkenederivatives, which are alkanes substituted with or linked to chemicalsubstituents such as nitro-, sulphur-, halogen- and oxygen-containinggroups, and/or aromatic groups.

The term “cofactor” means any non-protein substance that is necessary orbeneficial to the activity of an enzyme. A “coenzyme” means a cofactorthat interacts directly with and serves to promote a reaction catalyzedby an enzyme. Many coenzymes serve as carriers. For example, NAD+ andNADP+ carry hydrogen atoms from one enzyme to another. An “ancillaryprotein” means any protein substance that is necessary or beneficial tothe activity of an enzyme.

The terms “oxygen donor”, “oxidizing agent” and “oxidant” mean asubstance, molecule or compound which donates oxygen to a substrate inan oxidation reaction. Typically, the oxygen donor is reduced (acceptselectrons). Exemplary oxygen donors, which are not limiting, includemolecular oxygen or dioxygen (O₂) and peroxides, including alkylperoxides such as t-butyl peroxide, and most preferably hydrogenperoxide (H₂O₂). A peroxide is any compound having two oxygen atomsbound to each other by a single bond, i.e., dioxygen (O₂) has a doublebond between the oxygen atoms.

An “oxidation enzyme” is an enzyme that catalyzes one or more oxidationreactions, typically by adding, inserting, contributing or transferringoxygen from a source or donor to a substrate. Such enzymes are alsocalled oxidoreductases or redox enzymes, and encompasses oxygenases,hydrogenases or reductases, oxidases and peroxidases. An “oxidase” is anoxidation enzyme that catalyzes a reaction in which molecular oxygen(dioxygen or O₂) is reduced, for example by donating electrons to (orreceiving protons from) hydrogen.

A “luminescent” substance means any substance which produces detectableelectromagnetic radiation, or a change in electromagnetic radiation,most notably visible light, by any mechanism, including color change, UVabsorbance, fluorescence and phosphorescence. Preferably, a luminescentsubstance according to the invention produces a detectable color,fluorescence or UV absorbance. The term “chemiluminescent agent” meansany substance which enhances the detectability of a luminescent (e.g.,fluorescent) signal, for example by increasing the strength or lifetimeof the signal.

A “polynucleotide” or “nucleotide sequence” is a series of nucleotidebases (also called “nucleotides”) in DNA and RNA, and means any chain oftwo or more nucleotides. A nucleotide sequence typically carries geneticinformation, including the information used by cellular machinery tomake proteins and enzymes. These terms include double or single strandedgenomic and cDNA, RNA, any synthetic and genetically manipulatedpolynucleotide, and both sense and anti-sense polynucleotide (althoughonly sense stands are being represented herein). This includes single-and double-stranded molecules, i.e., DNA-DNA, DNA-RNA and RNA-RNAhybrids, as well as “protein nucleic acids” (PNA) formed by conjugatingbases to an amino acid backbone. This also includes nucleic acidscontaining modified bases, for example thio-uracil, thio-guanine andfluoro-uracil.

The polynucleotides herein may be flanked by natural regulatorysequences, or may be associated with heterologous sequences, includingpromoters, enhancers, response elements, signal sequences,polyadenylation sequences, introns, 5′- and 3′-non-coding regions, andthe like. The nucleic acids may also be modified by many means known inthe art. Non-limiting examples of such modifications includemethylation, “caps”, substitution of one or more of the naturallyoccurring nucleotides with an analog, and internucleotide modificationssuch as, for example, those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoroamidates, carbamates, etc.) andwith charged linkages (e.g., phosphorothioates, phosphorodithioates,etc.).

A “coding sequence” or a sequence “encoding” a polypeptide, protein orenzyme is a nucleotide sequence that, when expressed, results in theproduction of that polypeptide, protein or enzyme, i.e., the nucleotidesequence encodes an amino acid sequence for that polypeptide, protein orenzyme. A coding sequence is “under the control” of transcriptional andtranslational control sequences in a cell when RNA polymerasetranscribes the coding sequence into mRNA, which is then trans-RNAspliced and translated into the protein encoded by the coding sequence.Preferably, the coding sequence is a double-stranded DNA sequence whichis transcribed and translated into a polypeptide in a cell in vitro orin vivo when placed under the control of appropriate regulatorysequences. The boundaries of the coding sequence are determined by astart codon at the 5′ (amino) terminus and a translation stop codon atthe 3′ (carboxyl) terminus. A coding sequence can include, but is notlimited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomicDNA sequences from eukaryotic (e.g., mammalian) DNA, and even syntheticDNA sequences. If the coding sequence is intended for expression in aeukaryotic cell, a polyadenylation signal and transcription terminationsequence will usually be located 3′ to the coding sequence.

The term “gene”, also called a “structural gene” means a DNA sequencethat codes for or corresponds to a particular sequence of amino acidswhich comprise all or part of one or more proteins or enzymes, and mayor may not include regulatory DNA sequences, such as promoter sequences,which determine for example the conditions under which the gene isexpressed. Some genes, which are not structural genes, may betranscribed from DNA to RNA, but are not translated into an amino acidsequence. Other genes may function as regulators of structural genes oras regulators of DNA transcription. A gene encoding a protein of theinvention for use in an expression system, whether genomic DNA or cDNA,can be isolated from any source, particularly from a human cDNA orgenomic library. Methods for obtaining genes are well known in the art,e.g., Sambrook et al (supra).

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining this invention, thepromoter sequence is bounded at its 3′ terminus by the transcriptioninitiation site and extends upstream (5′ direction) to include theminimum number of bases or elements necessary to initiate transcriptionat levels detectable above background.

Polynucleotides are “hybridizable” to each other when at least onestrand of one polynucleotide can anneal to another polynucleotide underdefined stringency conditions. Stringency of hybridization isdetermined, e.g., by (a) the temperature at which hybridization and/orwashing is performed, and (b) the ionic strength and polarity (e.g.,formamide) of the hybridization and washing solutions, as well as otherparameters. Hybridization requires that the two polynucleotides containsubstantially complementary sequences; depending on the stringency ofhybridization, however, mismatches may be tolerated. Typically,hybridization of two sequences at high stringency (such as, for example,in an aqueous solution of 0.5×SSC at 65° C.) requires that the sequencesexhibit some high degree of complementarity over their entire sequence.Conditions of intermediate stringency (such as, for example, an aqueoussolution of 2×SSC at 65° C.) and low stringency (such as, for example,an aqueous solution of 2×SSC at 55° C.), require correspondingly lessoverall complementarity between the hybridizing sequences. (1×SSC is0.15 M NaCl, 0.015 M Na citrate.) Polynucleotides that hybridize includethose which anneal under suitable stringency conditions and which encodepolypeptides or enzymes having the same function, such as the ability tocatalyze an oxidation, oxygenase, or coupling reaction of the invention.

The term “expression system” means a host cell and compatible vectorunder suitable conditions, e.g. for the expression of a protein codedfor by foreign DNA carried by the vector and introduced to the hostcell. Common expression systems include bacteria (e.g., E. coli and B.subtilis) or yeast (e.g., S. cerevisiae) host cells and plasmid vectors,and insect host cells and Baculovirus vectors. As used herein, a “facileexpression system” means any expression system that is foreign orheterologous to a selected polynucleotide or polypeptide, and whichemploys host cells that can be grown or maintained more advantageouslythan cells that are native or heterologous to the selectedpolynucleotide or polypeptide, or which can produce the polypeptide moreefficiently or in higher yield. For example, the use of robustprokaryotic cells to express a protein of eukaryotic origin would be afacile expression system. Preferred facile expression systems include E.coli, B. subtilis and S. cerevisiae host cells and any suitable vector.

The term “transformation” means the introduction of a foreign (i.e.,extrinsic or extracellular) gene, DNA or RNA sequence to a host cell, sothat the host cell will express the introduced gene or sequence toproduce a desired substance, typically a protein or enzyme coded by theintroduced gene or sequence. The introduced gene or sequence may includeregulatory or control sequences, such as start, stop, promoter, signal,secretion, or other sequences used by the genetic machinery of the cell.A host cell that receives and expresses introduced DNA or RNA has been“transformed” and is a “transformant” or a “clone.” The DNA or RNAintroduced to a host cell can come from any source, including cells ofthe same genus or species as the host cell, or cells of a differentgenus or species.

The terms “vector”, “vector construct” and “expression vector” mean thevehicle by which a DNA or RNA sequence (e.g. a foreign gene) can beintroduced into a host cell, so as to transform the host and promoteexpression (e.g. transcription and translation) of the introducedsequence. Vectors typically comprise the DNA of a transmissible agent,into which foreign DNA encoding a protein is inserted by restrictionenzyme technology. A common type of vector is a “plasmid”, whichgenerally is a self-contained molecule of double-stranded DNA, that canreadily accept additional (foreign) DNA and which can readily introducedinto a suitable host cell. A large number of vectors, including plasmidand fungal vectors, have been described for replication and/orexpression in a variety of eukaryotic and prokaryotic hosts.Non-limiting examples include pKK plasmids (Clonetech), pUC plasmids,pET plasmids (Novagen, Inc., Madison, Wis.), pRSET or pREP plasmids(Invitrogen, San Diego, Calif.), or pMAL plasmids (New England Biolabs,Beverly, Mass.), and many appropriate host cells, using methodsdisclosed or cited herein or otherwise known to those skilled in therelevant art. Recombinant cloning vectors will often include one or morereplication systems for cloning or expression, one or more markers forselection in the host, e.g., antibiotic resistance, and one or moreexpression cassettes. Preferred vectors are described in the Examples,and include without limitations pcWori, pET-26b(+), pXTD14, pYEX-S1,pMAL, and pET22-b(+). Other vectors may be employed as desired by oneskilled in the art. Routine experimentation in biotechnology can be usedto determine which vectors are best suited for used with the invention,if different than as described in the Examples. In general, the choiceof vector depends on the size of the polynucleotide sequence and thehost cell to be employed in the methods of this invention.

The terms “express” and “expression” mean allowing or causing theinformation in a gene or DNA sequence to become manifest, for exampleproducing a protein by activating the cellular functions involved intranscription and translation of a corresponding gene or DNA sequence. ADNA sequence is expressed in or by a cell to form an “expressionproduct” such as a protein. The expression product itself, e.g. theresulting protein, may also be said to be “expressed” by the cell. Apolynucleotide or polypeptide is expressed recombinantly, for example,when it is expressed or produced in a foreign host cell under thecontrol of a foreign or native promoter, or in a native host cell underthe control of a foreign promoter.

A polynucleotide or polypeptide is “over-expressed” when it is expressedor produced in an amount or yield that is substantially higher than agiven base-line yield, e.g. a yield that occurs in nature. For example,a polypeptide is over-expressed when the yield is substantially greaterthan the normal, average or base-line yield of the nativepolypolypeptide in native host cells under given conditions, for exampleconditions suitable to the life cycle of the native host cells.

“Isolation” or “purification” of a polypeptide or enzyme refers to thederivation of the polypeptide by removing it from its originalenvironment (for example, from its natural environment if it isnaturally occurring, or form the host cell if it is produced byrecombinant DNA methods). Methods for polypeptide purification arewell-known in the art, including, without limitation, preparativedisc-gel electrophoresis, isoelectric focusing, HPLC, reversed-phaseHPLC, gel filtration, ion exchange and partition chromatography, andcountercurrent distribution. For some purposes, it is preferable toproduce the polypeptide in a recombinant system in which the proteincontains an additional sequence tag that facilitates purification, suchas, but not limited to, a polyhistidine sequence. The polypeptide canthen be purified from a crude lysate of the host cell by chromatographyon an appropriate solid-phase matrix. Alternatively, antibodies producedagainst the protein or against peptides derived therefrom can be used aspurification reagents. Other purification methods are possible. Apurified polynucleotide or polypeptide may contain less than about 50%,preferably less than about 75%, and most preferably less than about 90%,of the cellular components with which it was originally associated. A“substantially pure” enzyme indicates the highest degree of purity whichcan be achieved using conventional purification techniques known in theart.

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1-67. (canceled)
 68. An isolated polypeptide having activity of acytochrome P450 variant, wherein the variant is characterized ascomprising a higher capability than the corresponding wild-typecytochrome P450 to oxidize at least one substrate selected from analkane comprising a carbon-chain of no more than 8 carbons and an alkenecomprising a carbon-chain no more than 8 carbons, and wherein thewild-type cytochrome P450 comprises an amino acid sequence having atleast 90% sequence identity to SEQ ID NO: 2, and the cytochrome P450variant comprises an amino acid substitution at two residues of SEQ IDNO:2 selected from the group consisting of V78, T175, A184, H236, E252,R255, A290, and L353.
 69. The isolated polypeptide of claim 68, whereinthe amino acid sequence has at least 95% sequence identity to SEQ ID NO:2.
 70. The isolated polypeptide of claim 69, wherein the amino acidsequence is SEQ ID NO:
 2. 71. The isolated polypeptide of claim 68,wherein the higher capability is a higher maximum turnover rate of thesubstrate into an oxidized product.
 72. The isolated polypeptide ofclaim 71, wherein the maximum turnover rate of the variant is at least5-10 times the maximum turnover rate of the wild-type.
 73. The isolatedpolypeptide of claim 71, wherein the substrate is an optionallysubstituted alkane and the capability to oxidize is the capability tohydroxylate.
 74. The isolated polypeptide of claim 71, wherein thesubstrate is an optionally substituted alkene and the capability tooxidize is the capability to epoxidize alkene bonds.
 75. The isolatedpolypeptide of claim 68, wherein the cytochrome P450 variant comprisesat least three amino acid substitutions selected from the groupconsisting of V78, T175, A184, H236, E252, R255, A290 and L353.
 76. Theisolated polypeptide of claim 75, wherein the cytochrome P450 variantcomprises at least four amino acid substitutions selected from the groupconsisting of V78, T175, A184, H236, E252, R255, A290 and L353.
 77. Theisolated polypeptide of claim 76, wherein the cytochrome P450 variantcomprises at least five amino acid substitutions selected from the groupconsisting of V78, T175, A184, H236, E252, R255, A290 and L353.
 78. Theisolated polypeptide of claim 77, wherein the cytochrome P450 variantcomprises at least six amino acid substitutions selected from the groupconsisting of V78, T175, A184, H236, E252, R255, A290 and L353.
 79. Theisolated polypeptide of claim 78, wherein the cytochrome P450 variantcomprises at least seven amino acid substitutions selected from thegroup consisting of V78, T175, A184, H236, E252, R255, A290 and L353.80. The isolated polypeptide of claim 79, wherein the cytochrome P450variant comprises amino acid substitutions at V78, T175, A184, H236,E252, R255, A290 and L353.
 81. The isolated polypeptide of claim 68,wherein the amino acid substitutions are selected from V78A, T175I,A184V, H236Q, E252G, R255S, A290V, and L353V.
 82. The isolatedpolypeptide of claim 68, wherein the cytochrome P450 variant comprisesat least one further amino acid substitution at a residue selected fromF87, N186, F205, D217, S226, T235, G396, R471, E494 and S1024.
 83. Theisolated polypeptide of claim 80, wherein the at least one further aminoacid substitution is selected from F87A, N186D, F205C, D217V, S226I,S226R, T235A, G396M, R471A, E494K, and S1024E.